Self-contained biological analysis

ABSTRACT

Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analyses include nucleic acid amplification and detection and immuno-PCR.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.application Ser. No. 16/045,064, filed Jul. 25, 2018, which is adivisional of, and claims priority to, U.S. application Ser. No.14/569,227, filed Dec. 12, 2014 and issued as U.S. Pat. No. 10,058,868on Aug. 28, 2018, which is a divisional of, and claims priority to, U.S.application Ser. No. 13/765,249, filed Feb. 12, 2013 and issued as U.S.Pat. No. 8,940,526 on Jan. 27, 2015, which is a continuation of, andclaims priority to, U.S. application Ser. No. 11/913,120, filed Sep. 16,2009 and issued as U.S. Pat. No. 8,394,608 on Mar. 12, 2013, which is anational stage application of PCT Application Serial No.PCT/US2006/017665, filed May 8, 2006, which claims priority to U.S.Application Ser. No. 60/679,052, filed on May 9, 2005, the entiredisclosures of each of which are incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with government support under Grant Nos. U01AI061611 and R43 AI063695 awarded by National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms and the low levels of organism present in somepathological specimens. It is often impractical to run large panels ofPCR assays, one for each possible causative organism, most of which areexpected to be negative. The problem is exacerbated when pathogennucleic acid is at low concentration and requires a large volume ofsample to gather adequate reaction templates. In some cases there isinadequate sample to assay for all possible etiological agents. Asolution is to run “multiplex PCR” wherein the sample is concurrentlyassayed for multiple targets in a single reaction. While multiplex PCRhas proved to be valuable in some systems, shortcomings exist concerningrobustness of high level multiplex reactions and difficulties for clearanalysis of multiple products. To solve these problems, the assay may besubsequently divided into multiple secondary PCRs. Nesting secondaryreactions within the primary product increases robustness. However, thisfurther handling can be expensive and may lead to contamination or otherproblems.

Similarly, immuno-PCR (“iPCR”) has the potential for sensitive detectionof a wide variety of antigens. However, because traditional ELISAtechniques have been applied to iPCR, iPCR often suffers fromcontamination issues that are problematic using a PCR-based detectionmethod.

The present invention addresses various issues of contamination inbiological analysis.

SUMMARY OF THE INVENTION

Accordingly, a rapid, sensitive assay that simultaneously assays formultiple biological substances, including organisms, is provided. Theself-contained system illustratively employs an inexpensive disposableplastic pouch in a self-contained format, allowing for nested PCR andother means to identify bio-molecules, illustratively while minimizingcontamination and providing for robust amplification.

Thus, in one aspect of the present invention a container for performingtwo-stage amplification on a sample in a closed system is provided, thecontainer comprising a first-stage reaction zone comprising afirst-stage reaction blister configured for first-stage amplification ofthe sample, an additional reservoir fluidly connected to the first-stagereaction blister, the additional reservoir configured for providingadditional fluids to the sample, and a second-stage reaction zonefluidly connected to the first-stage reaction zone, the second-stagereaction zone comprising a plurality of second-stage reaction chambers,each second-stage reaction chamber comprising a pair of primersconfigured for further amplification of the sample. In one illustrativeexample, the first-stage reaction zone is a first-stage PCRamplification zone. In another illustrative example, the first stagereaction zone is an antigen-binding zone for immuno-PCR, in whichantigens present in the sample are recognized and associated with aparticular nucleic acid segment and the second stage reaction zone is anucleic acid amplification zone. In yet another illustrative example,the container further comprises a cell lysis zone comprising particlesfor lysing cells or spores located in the sample, and a nucleic acidpreparation zone comprising components for purifying nucleic acids.Illustratively, the blisters comprise a flexible material, such thatpressure provided on an individual blister collapses the blister,forcing the contents from the blister.

In another aspect of the present invention, a container is providedcomprising a flexible portion having a plurality of blisters fluidlyconnected via a plurality of channels, and a plurality of reservoirs,each reservoir containing a reaction component, and each reservoirfluidly connected to at least one of the plurality of blisters, and asealable port configured for receiving the sample the sealable portfluidly connected to one of the plurality of blisters. In oneillustrative embodiment, the reaction components are in dried form, andthe container further comprises a second sealable port fluidly connectedto each of the plurality of reservoirs, the port configured forreceiving water to rehydrate the reaction components.

In a further aspect of the present invention, a method for lysing cellsin a sample is provided, the method comprising providing a flexiblecontainer comprising a cell lysis blister, introducing cells into thecell lysis blister, and applying force to the blister to move theparticles and sample to generate high velocity impacts resulting in alysate.

In yet another aspect of the present invention, a device for analyzing asample for the presence of nucleic acids is provided, the deviceconfigured to receive a container of the present invention therein, aplurality of actuators positioned corresponding to various blisters ofthe container, each actuator configured to apply pressure to thecorresponding blister of the container, a first heater/cooler deviceconfigured for thermal cycling the contents of one of the blisters, anda second heater/cooler device for thermal cycling the second-stagechamber.

In still another aspect of the present invention, methods are provided.In one illustrative method, nucleic acids are amplified. In anotherillustrative method, antigens are detected using immuno-PCR.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flexible pouch according to one embodiment of thisinvention.

FIG. 2 shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1 .

FIG. 2 a shows an embodiment of a portion of a bladder corresponding tothe cell lysis zone shown in FIG. 2 .

FIG. 2 b shows an embodiment of the cell lysis zone of the flexiblepouch according to FIG. 1 having an alternative vortexing mechanism.

FIG. 3 shows an embodiment of the nucleic acid preparation zone of theflexible pouch according to FIG. 1 .

FIG. 4 shows an embodiment of the first-stage amplification zone of theflexible pouch according to FIG. 1 .

FIG. 5 is similar to FIG. 1 , except showing an alternative embodimentof a pouch.

FIG. 5 a is a cross-sectional view of the fitment of the pouch of FIG. 5.

FIG. 5 b is an enlargement of a portion of the pouch of FIG. 5 .

FIG. 6 is a perspective view of another alternative embodiment of apouch.

FIG. 6 a is a cross-sectional view of the fitment of the pouch of FIG. 6.

FIG. 7 shows illustrative bladder components for use with the pouch ofFIG. 6 .

FIG. 8 is an exploded perspective view of an instrument for use with thepouch of FIG. 6 , including the pouch of FIG. 6 .

FIG. 9 shows a partial cross-sectional view of the instrument of FIG. 8, including the bladder components of FIG. 7 , with the pouch of FIG. 6shown in shadow.

FIG. 10 shows a partial cross-sectional view of the instrument of FIG. 8, including various bladders for pinch valves and the pouch of FIG. 6 .

FIG. 11 shows schemes for ELISA and immuno-PCR, secondary antibody (A);capture antibody (C); enzyme (E); reporter antibody (R); bi-functionalbinding moiety (S) and antigen (T).

FIG. 12 is similar to FIG. 6 , except showing a pouch configured forimmuno-PCR.

FIG. 13 is similar to FIG. 6 , except showing a pouch configured forboth PCR and immuno-PCR.

FIG. 14 shows amplification curves from second-stage amplification of asample that was lysed and amplified in a pouch of FIG. 5 ( - - -positive control; - - - S. cerevisaie target 1; - S. cerevisaie target2; - S. cerevisaie target 3; - - - - - - S. pombe target 1; - - - - S.pombe target 2; - - - - negative controls).

DETAILED DESCRIPTION

The self-contained nucleic acid analysis pouches described herein may beused to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. In one embodiment, the pouchis used to assay for multiple pathogens. Illustratively, various stepsmay be performed in the optionally disposable pouch, including nucleicacid preparation, primary large volume multiplex PCR, dilution ofprimary amplification product, and secondary PCR, culminating withreal-time detection and/or post-amplification analysis such asmelting-curve analysis. It is understood, however, that pathogendetection is one exemplary use and the pouches may be used for othernucleic acid analysis or detection of other substances, including butnot limited to peptides, toxins, and small molecules. Further, it isunderstood that while the various steps may be performed in pouches ofthe present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

FIG. 1 shows an illustrative self-contained nucleic acid analysis pouch10. Pouch 10 has a cell lysis zone 20, a nucleic acid preparation zone40, a first-stage amplification zone 60, and a second-stageamplification zone 80. A sample containing nucleic acid is introducedinto the pouch 10 via sample injection port 12. Pouch 10 comprises avariety of channels and blisters of various sizes and is arranged suchthat the sample flows through the system. The sample passes through thevarious zones and is processed accordingly.

Sample processing occurs in various blisters located within pouch 10.Various channels are provided to move the sample within and betweenprocessing zones, while other channels are provided to deliver fluidsand reagents to the sample or to remove such fluids and reagents fromthe sample. Liquid within pouch 10 illustratively is moved betweenblisters by pressure, illustratively pneumatic pressure, as describedbelow, although other methods of moving material within the pouch arecontemplated.

While other containers may be used, illustratively, pouch 10 is formedof two layers of a flexible plastic film or other flexible material suchas polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene, polymethylmethacrylate, and mixtures thereof that can bemade by any process known in the art, including extrusion, plasmadeposition, and lamination. Metal foils or plastics with aluminumlamination also may be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing. Illustratively, the material has low nucleic acidbinding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by tryingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in the blisters 82 of the second stageamplification zone 80 of pouch 10, then one or both layers at blisters82 would be left without the foil. In the example of PCR, film laminatescomposed of polyester (Mylar, Dupont, Wilmington Del.) of about 0.0048inch (0.1219 mm) thick and polypropylene films of 0.001-0.003 inch(0.025-0.076 mm) thick perform well. Illustratively, pouch 10 is made ofa clear material capable of transmitting approximately 80%-90% ofincident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpneumatic pressure, the pouch material illustratively is flexible enoughto allow the pneumatic pressure to have the desired effect. The term“flexible” is herein used to describe a physical characteristic of thematerial of pouch. The term “flexible” is herein defined as readilydeformable by the levels of pneumatic pressure used herein withoutcracking, breaking, crazing, or the like. For example, thin plasticsheets, such as Saran™ wrap and Ziploc® bags, as well as thin metalfoil, such as aluminum foil, are flexible. However, only certain regionsof the blisters and channels need be flexible, even in embodimentsemploying pneumatic pressure. Further, only one side of the blisters andchannels need to be flexible, as long as the blisters and channels arereadily deformable. Other regions of the pouch 10 may be made of a rigidmaterial or may be reinforced with a rigid material.

Illustratively, a plastic film is used for pouch 10. A sheet of metal,illustratively aluminum, or other suitable material, may be milled orotherwise cut, to create a die having a pattern of raised surfaces. Whenfitted into a pneumatic press (illustratively A-5302-PDS, JanesvilleTool Inc., Milton Wis.), illustratively regulated at an operatingtemperature of 195° C., the pneumatic press works like a printing press,melting the sealing surfaces of plastic film only where the die contactsthe film. Various components, such as PCR primers (illustrativelyspotted onto the film and dried), antigen binding substrates, magneticbeads, and zirconium silicate beads may be sealed inside variousblisters as the pouch 10 is formed. Reagents for sample processing canbe spotted onto the film prior to sealing, either collectively orseparately. In one embodiment, nucleotide tri-phosphates (NTPs) arespotted onto the film separately from polymerase and primers,essentially eliminating activity of the polymerase until the reaction ishydrated by an aqueous sample. If the aqueous sample has been heatedprior to hydration, this creates the conditions for a true hot-start PCRand reduces or eliminates the need for expensive chemical hot-startcomponents. This separate spotting is discussed further below, withrespect to FIG. 5 b , but it is understood that such spotting may beused with any of the embodiments discussed herein.

When pneumatic pressure is used to move materials within pouch 10, inone embodiment a “bladder” may be employed. The bladder assembly 710, aportion of which is shown in FIG. 2 a , may be manufactured in a processsimilar to that of making the pouch, but individual blisters in thebladder assembly 710 include pneumatic fittings (illustratively fitting724 a) allowing individual bladders within the bladder assembly 710 tobe pressurized by a compressed gas source. Because the bladder assemblyis subjected to compressed gas and may be used multiple times, thebladder assembly may be made from tougher or thicker material than thepouch.

When pouch 10 is placed within the instrument, the pneumatic bladderassembly 710 is pressed against one face of the pouch 10, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 10. In addition to pneumaticbladders corresponding to many of the blisters of pouch 10, the bladderassembly may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch10. When activated, these additional pneumatic actuators form pinchvalves to pinch off and close the corresponding channels. To confineliquid within a particular blister of pouch 10, the pinch valvepneumatic actuators are inflated over the channels leading to and fromthe blister, such that the actuators function as pinch valves to pinchthe channels shut. Illustratively, to mix two volumes of liquid indifferent blisters, the pinch valve pneumatic actuator sealing theconnecting channel is depressurized, and the pneumatic bladders over theblisters are alternately pressurized, forcing the liquid back and forththrough the channel connecting the blisters to mix the liquid therein.The pinch valve pneumatic actuators may be of various shapes and sizesand may be configured to pinch off more than one channel at a time. Suchan illustrative pinch valve is illustrated in FIG. 1 as pinch valve 16,which may be used to close all injection ports. While pneumaticactuators are discussed herein, it is understood that other ways ofproviding pressure to the pouch are contemplated, including variouselectromechanical actuators such as linear stepper motors, motor-drivencams, rigid paddles driven by pneumatic, hydraulic or electromagneticforces, rollers, rocker-arms, and in some cases, cocked springs. Inaddition, there are a variety of methods of reversibly or irreversiblyclosing channels in addition to applying pressure normal to the axis ofthe channel. These include kinking the bag across the channel,heat-sealing, rolling an actuator, and a variety of physical valvessealed into the channel such as butterfly valves and ball valves.Additionally, small Peltier devices or other temperature regulators maybe placed adjacent the channels and set at a temperature sufficient tofreeze the fluid, effectively forming a seal. Also, while the design ofFIG. 1 is adapted for an automated instrument featuring actuatorelements positioned over each of the blisters and channels, it is alsocontemplated that the actuators could remain stationary, and the pouchcould be transitioned in one or two dimensions such that a small numberof actuators could be used for several of the processing stationsincluding sample disruption, nucleic-acid capture, first andsecond-stage PCR, and other applications of the pouch such asimmuno-assay and immuno-PCR. Rollers acting on channels and blisterscould prove particularly useful in a configuration in which the pouch istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

With reference to FIG. 1 , an illustrative sample pouch 10 configuredfor nucleic acid extraction and multiplex PCR is provided. The sampleenters pouch 10 via sample injection port 12 in fitment 90. Injectorport 12 may be a frangible seal, a one-way valve, or other entry port.Vacuum from inside pouch 10 may be used to draw the sample into pouch10, a syringe or other pressure may be used to force the sample intopouch 10, or other means of introducing the sample into pouch 10 viainjector port 12 may be used. The sample travels via channel 14 to thethree-lobed blister 22 of the cell lysis zone 20, wherein cells in thesample are lysed. Once the sample enters three-lobed blister 22, pinchvalve 16 is closed. Along with pinch valve 36, which may have beenalready closed, the closure of pinch valve 16 seals the sample inthree-lobed blister 22. It is understood that cell lysis may not benecessary with every sample. For such samples, the cell lysis zone maybe omitted or the sample may be moved directly to the next zone.However, with many samples, cell lysis is needed. In one embodiment,bead-milling is used to lyse the cells.

Bead-milling, by shaking or vortexing the sample in the presence oflysing particles such as zirconium silicate (ZS) beads 34, is aneffective method to form a lysate. It is understood that, as usedherein, terms such as “lyse,” “lysing,” and “lysate” are not limited torupturing cells, but that such terms include disruption of non-cellularparticles, such as viruses. FIG. 2 displays one embodiment of a celllysis zone 20, where convergent flow creates high velocity bead impacts,to create lysate. Illustratively, the two lower lobes 24, 26 ofthree-lobed blister 22 are connected via channel 30, and the upper lobe28 is connected to the lower lobes 24, 26 at the opposing side 31 ofchannel 30. FIG. 2 a shows a counterpart portion of the bladder assembly710 that would be in contact with the cell lysis zone 20 of the pouch10. When pouch 10 is placed in an instrument, adjacent each lobe 24, 26,28 on pouch 10 is a corresponding pneumatic bladder 724, 726, 728 in thebladder assembly 710. It is understood that the term “adjacent,” whenreferring to the relationship between a blister or channel in a pouchand its corresponding pneumatic actuator, refers to the relationshipbetween the blister or channel and the corresponding pneumatic actuatorwhen the pouch is placed into the instrument. In one embodiment, thepneumatic fittings 724 a, 726 a of the two lower pneumatic bladders 724,726 adjacent lower lobes 24, 26 are plumbed together. The pneumaticfittings 724 a, 726 a and the pneumatic fitting 728 a of upper pneumaticbladder 728 adjacent upper lobe 28 are plumbed to the opposing side ofan electrically actuated valve configured to drive a double-actingpneumatic cylinder. Thus configured, pressure is alternated between theupper pneumatic bladder 728 and the two lower pneumatic bladders 724,726. When the valve is switched back and forth, liquid in pouch 10 isdriven between the lower lobes 24, 26 and the upper lobe 28 through anarrow nexus 32 in channel 30. As the two lower lobes 24, 26 arepressurized at the same time, the flow converges and shoots into theupper lobe 28. Depending on the geometry of the lobes, the collisionvelocity of beads 34 at the nexus 32 may be at least about 12 m/sec,providing high-impact collisions resulting in lysis. The illustrativethree-lobed system allows for good cell disruption and structuralrobustness, while minimizing size and pneumatic gas consumption. WhileZS beads are used as the lysing particles, it is understood that thischoice is illustrative only, and that other materials and particles ofother shapes may be used. It is also understood that otherconfigurations for cell lysis zone 20 are within the scope of thisinvention.

While a three-lobed blister is used for cell lysis, it is understoodthat other multi-lobed configurations are within the scope of thisinvention. For instance, a four-lobed blister, illustratively in acloverleaf pattern, could be used, wherein the opposite blisters arepressurized at the same time, forcing the lysing particles toward eachother, and then angling off to the other two lobes, which then may bepressurized together. Such a four-lobed blister would have the advantageof having high-velocity impacts in both directions. Further, it iscontemplated that single-lobed blisters may be used, wherein the lysingparticles are moved rapidly from one portion of the single-lobed blisterto the other. For example, pneumatic actuators may be used to close offareas of the single-lobed blister, temporarily forming a multi-lobedblister in the remaining areas. It may also be possible to move thesample and lysing particles quickly enough to effect lysis within asingle-lobed lysis blister without temporarily forming a multi-lobedblister. In one such alternative embodiment, as shown in FIG. 2 b ,vortexing may be achieved by impacting the pouch with rotating blades orpaddles 21 attached to an electric motor 19. The blades 21 may impactthe pouch at the lysis blister or may impact the pouch near the lysisblister, illustratively at an edge 17 adjacent the lysis blister. Insuch an embodiment, the lysis blister may comprise one or more blisters.Other actuation methods may also be used such as motor, pneumatic,hydraulic, or electromagnetically-driven paddles acting on the lobes ofthe device. Rollers or rotary paddles can be used to drive fluidtogether at the nexus 32 of FIG. 2 , illustratively if a recirculationmeans is provided between the upper and lower lobes and the actuatorprovides peristaltic pumping action. Other configurations are within thescope of this invention.

FIG. 2 a also shows pneumatic bladder 716 with pneumatic fitting 716 a,and pneumatic bladder 736 with pneumatic fitting 736 a. When the pouch10 is placed in contact with bladder assembly 710, bladder 716 lines upwith channel 12 to complete pinch valve 16. Similarly, bladder 736 linesup with channel 38 to complete pinch valve 36. Operation of pneumaticbladders 716 and 736 allow pinch valves 16 and 36 to be opened andclosed. While only the portion of bladder assembly 710 adjacent the celllysis zone is shown, it is understood that bladder assembly 710 would beprovided with similar arrangements of pneumatic blisters to control themovement of fluids throughout the remaining zones of pouch 10.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758 and 6,780,617, andco-pending U.S. patent application Ser. No. 10/478,453, hereinincorporated by reference. However, including the cell lysis within thesealed PCR vessel can improve ease of use and safety, particularly ifthe sample to be tested may contain a biohazard. In the embodimentsillustrated herein, the waste from cell lysis, as well as all othersteps, remains within the sealed pouch. However, it is understood thatthe pouch contents could be removed for further testing.

Once the cells are lysed, pinch valve 36 is opened and the lysate ismoved through channel 38 to the nucleic acid preparation zone 40, asbest seen in FIG. 3 , after which, pinch valve 36 is closed, sealing thesample in nucleic acid preparation zone 40. In the embodimentillustrated in FIG. 3 , purification of nucleic acids takes thebead-milled material and uses affinity binding to silica-basedmagnetic-beads 56, washing the beads with ethanol, and eluting thenucleic acids with water or other fluid, to purify the nucleic acid fromthe cell lysate. The individual components needed for nucleic acidextraction illustratively reside in blisters 44, 46, 48, which areconnected by channels 45, 47, 49 to allow reagent mixing. The lysateenters blister 44 from channel 38. Blister 44 may be provided withmagnetic beads 56 and a suitable binding buffer, illustratively ahigh-salt buffer such as that of 1-2-3 Sample Preparation Kit (IdahoTechnology, Salt Lake City, Utah) or either or both of these componentsmay be provided subsequently through one or more channels connected toblister 44. The nucleic acids are captured on beads 56, pinch valve 53is then opened, and the lysate and beads 56 may be mixed by gentlepressure alternately on blisters 44 and 58 and then moved to blister 58via pneumatic pressure illustratively provided by a correspondingpneumatic bladder on bladder assembly 710. The magnetic beads 56 arecaptured in blister 58 by a retractable magnet 50, which is located inthe instrument adjacent blister 58, and waste may be moved to a wastereservoir or may be returned to blister 44 by applying pressure toblister 58. Pinch valve 53 is then closed. The magnetic beads 56 arewashed with ethanol, isopropanol, or other organic or inorganic washsolution provided from blister 46, upon release of pinch valve 55.Optionally, magnet 50 may be retracted allowing the beads to be washedby providing alternate pressure on blisters 46 and 58. The beads 56 areonce again captured in blister 58 by magnet 50, and the non-nucleic acidportion of the lysate is washed from the beads 56 and may be moved backto blister 46 and secured by pinch valve 55 or may be washed away viaanother channel to a waste reservoir. Once the magnetic beads arewashed, pinch valve 57 is opened, releasing water (illustrativelybuffered water) or another nucleic acid eluant from blister 48. Onceagain, the magnet 50 may be retracted to allow maximum mixing of waterand beads 56, illustratively by providing alternate pressure on blisters48 and 58. The magnet 50 is once again deployed to collect beads 56.Pinch valve 59 is released and the eluted nucleic acid is moved viachannel 52 to first-stage amplification zone 60. Pinch valve 59 is thenclosed, thus securing the sample in first-stage amplification zone 60.

It is understood that the configuration for the nucleic acid preparationzone 40, as shown in FIG. 3 and described above, is illustrative only,and that various other configurations are possible within the scope ofthe present disclosure.

The ethanol, water, and other fluids used herein may be provided to theblisters in various ways. The fluids may be stored in the blisters, thenecks of which may be pinched off by various pinch valves or frangibleportions that may be opened at the proper time in the sample preparationsequence. Alternatively, fluid may be stored in reservoirs in the pouchas shown pouch 110 in FIG. 5 , or in the fitment as discussed withrespect to pouch 210 of FIG. 6 , and moved via channels, as necessary.In still another embodiment, the fluids may be introduced from anexternal source, as shown in FIG. 1 , especially with respect to ethanolinjection ports 41, 88 and plungers 67, 68, 69. Illustratively, plungers67, 68, 69 may inserted into fitment 90, illustratively of a more rigidmaterial, and may provide a measured volume of fluid upon activation ofthe plunger, as in U.S. patent application Ser. No. 10/512,255, hereinincorporated by reference. The measured volume may be the same ordifferent for each of the plungers. Finally, in yet another embodiment,the pouch may be provided with a measured volume of the fluid that isstored in one or more blisters, wherein the fluid is contained withinthe blister, illustratively provided in a small sealed pouch within theblister, effectively forming a blister within the blister. At theappropriate time, the sealed pouch may then be ruptured, illustrativelyby pneumatic pressure, thereby releasing the fluid into the blister ofthe pouch. The instrument may also be configured the provide some or allof the reagents directly through liquid contacts between the instrumentand the fitment or pouch material provided that the passage of fluid istightly regulated by a one-way valve to prevent the instrument frombecoming contaminated during a run. Further, it will often be desirablefor the pouch or its fitment to be sealed after operation to prohibitcontaminating DNA to escape from the pouch. Various means are known toprovide reagents on demand such as syringe pumps, and to make temporaryfluid contact with the fitment or pouch, such as barbed fittings oro-ring seals. It is understood that any of these methods of introducingfluids to the appropriate blister may be used with any of theembodiments of the pouch as discussed herein, as may be dictated by theneeds of a particular application.

As discussed above, nested PCR involves target amplification performedin two stages. In the first-stage, targets are amplified, illustrativelyfrom genomic or reverse-transcribed template. The first-stageamplification may be terminated prior to plateau phase, if desired. Inthe secondary reaction, the first-stage amplicons may be diluted and asecondary amplification uses the same primers or illustratively usesnested primers hybridizing internally to the primers of the first-stageproduct. Advantages of nested PCR include: a) the initial reactionproduct forms a homogeneous and specific template assuring high fidelityin the secondary reaction, wherein even a relatively low-efficiencyfirst-stage reaction creates adequate template to support robustsecond-stage reaction; b) nonspecific products from the first-stagereaction do not significantly interfere with the second stage reaction,as different nested primers are used and the original amplificationtemplate (illustratively genomic DNA or reverse-transcription product)may be diluted to a degree that eliminates its significance in thesecondary amplification; and c) nested PCR enables higher-order reactionmultiplexing. First-stage reactions can include primers for severalunique amplification products. These products are then identified in thesecond-stage reactions. However, it is understood that first-stagemultiplex and second-stage singleplex is illustrative only and thatother configurations are possible. For example, the first-stage mayamplify a variety of different related amplicons using a single pair ofprimers, and second-stage may be used to target differences between theamplicons, illustratively using melting curve analysis.

Turning back to FIG. 1 , the nucleic acid sample enters the first-stageamplification zone 60 via channel 52 and is delivered to blister 61. APCR mixture, including a polymerase (illustratively a Taq polymerase),dNTPs, and primers, illustratively a plurality of pairs of primers formultiplex amplification, may be provided in blister 61 or may beintroduced into blister 61 via various means, as discussed above.Alternatively, dried reagents may be spotted onto the location ofblister 61 upon assembly of pouch 10, and water or buffer may beintroduced to blister 61, illustratively via plunger 68, as shown inFIG. 1 . As best seen in FIG. 4 , the sample is now secured in blister61 by pinch valves 59 and 72, and is thermocycled between two or moretemperatures, illustratively by heat blocks or Peltier devices that arelocated in the instrument and configured to contact blister 61. However,it is understood that other means of heating and cooling the samplecontained within blister 61, as are known in the art, are within thescope of this invention. Non-limiting examples of alternativeheating/cooling devices for thermal cycling include having a air-cycledblister within the bladder, in which the air in the pneumatic blisteradjacent blister 61 is cycled between two or more temperatures; ormoving the sample to temperature zones within the blister 61,illustratively using a plurality of pneumatic presses, as in U.S. patentapplication Ser. No. 10/478,453, herein incorporated by reference, or bytranslating pouch 10 on an axis or providing pouch 10 with a rotarylayout and spinning pouch 10 to move the contents between heat zones offixed temperature.

Nucleic acids from pathogens are often co-isolated with considerablequantities of host nucleic acids. These host-derived nucleic acids ofteninteract with primers, resulting in amplification of undesired productsthat then scavenge primers, dNTPs, and polymerase activity, potentiallystarving a desired product of resources. Nucleic acids from pathogenicorganisms are generally of low abundance, and undesired product is apotential problem. The number of cycles in the first-stage reaction ofzone 60 may be optimized to maximize specific products and minimizenon-specific products. It is expected that the optimum number of cycleswill be between about 10 to about 30 cycles, illustratively betweenabout 15 to about 20 cycles, but it is understood that the number ofcycles may vary depending on the particular target, host, and primersequence.

Following the first-stage multiplex amplification, the first-stageamplification product is diluted, illustratively in incomplete PCRmaster mix, before fluidic transfer to secondary reaction sites.

FIG. 4 shows an illustrative embodiment for diluting the sample in threesteps. In the first step, pinch valve 72 is opened and the sampleundergoes a two-fold dilution by mixing the sample in blister 61 with anequal volume of water or buffer from blister 62, which is provided toblister 62, as well as blisters 64 and 66, as discussed above. Squeezingthe volume back and forth between blisters 61, 62 provides thoroughmixing. As above, mixing may be provided by pneumatic bladders providedin the bladder 710 and located adjacent blisters 61, 62. The pneumaticbladders may be alternately pressurized, forcing the liquid back andforth. During mixing, a pinch valve 74 prevents the flow of liquid intothe adjacent blisters. At the conclusion of mixing, a volume of thediluted sample is captured in region 70, and pinch valve 72 is closed,sealing the diluted sample in region 70. Pinch valve 74 is opened andthe sample is further diluted by water or buffer provided in either orboth of blisters 63, 64. As above, squeezing the volume back and forthbetween blisters 63, 64 provides mixing. Subsequently, pinch valve 74 isclosed, sealing a further diluted volume of sample in region 71. Finaldilution takes place illustratively by using buffer or water provided ineither or both of blisters 65, 66, with mixing as above. Illustrativelythis final dilution takes place using an incomplete PCR master mix(e.g., containing all PCR reagents except primers) as the fluid.Optional heating of the contents of blister 66 prior to second-stageamplification can provide the benefits of hot-start amplificationwithout the need for expensive antibodies or enzymes. It is understood,however, that water or other buffer may be used for the final dilution,with additional PCR components provided in second-stage amplificationzone 80. While the illustrative embodiment uses three dilution stages,it is understood that any number of dilution stages may be used, toprovide a suitable level of dilution. It is also understood that theamount of dilution can be controlled by adjusting the volume of thesample captured in regions 70 and 71, wherein the smaller the amount ofsample captured in regions 70 and 71, the greater the amount of dilutionor wherein additional aliquots captured in region 70 and/or region 71 byrepeatedly opening and closing pinch valves 72 and 74 and/or pinchvalves 74 and 76 may be used to decrease the amount of dilution. It isexpected that about 10⁻² to about 10⁻⁵ dilution would be suitable formany applications.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 10 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

Subsequent to first-stage PCR and dilution, channel 78 transfers thesample to a plurality of low volume blisters 82 for secondary nestedPCR. In one illustrative embodiment, dried primers provided in thesecond-stage blisters are resuspended by the incoming aqueous materialto complete the reaction mixture. Optionally, fluorescent dyes such asLCGreen® Plus (Idaho Technology, Salt Lake City, Utah) used fordetection of double-stranded nucleic acid may be provided in eachblister or may be added to the incomplete PCR master mix provided at theend of the serial dilution, although it is understood that LCGreen® Plusis illustrative only and that other dyes are available, as are known inthe art. In another optional embodiment, dried fluorescently labeledoligonucleotide probes configured for each specific amplicon may beprovided in each respective second-stage blister, along with therespective dried primers. Further, while pouch 10 is designed to containall reactions and manipulations within, to reduce contamination, in somecircumstances it may be desirable to remove the amplification productsfrom each blister 82 to do further analysis. Other means for detectionof the second-stage amplicon, as are known in the art, are within thescope of this invention. Once the sample is transferred to blisters 82,pinch valves 84 and 86 are activated to close off blisters 82. Eachblister 82 now contains all reagents needed for amplification of aparticular target. Illustratively, each blister may contain a uniquepair of primers, or a plurality of blisters 82 may contain the sameprimers to provide a number of replicate amplifications.

It is noted that the embodiments disclosed herein use blisters for thesecond-stage amplification, wherein the blisters are formed of the sameor similar plastic film as the rest of the flexible portion. However, inmany embodiments, the contents of the second-stage blisters are neverremoved from the second-stage blisters, and, therefore, there is no needfor the second-stage reaction to take place in flexible blisters. It isunderstood that the second-stage reaction may take place in a pluralityof rigid, semi-rigid, or flexible chambers that are fluidly connected tothe blisters. The chambers could be sealed as in the present example byplacing pressure on flexible channels that connect the chambers, or maybe sealed in other ways, illustratively by heat sealing or use ofone-way valves. Various embodiments discussed herein include blistersprovided solely for the collection of waste. Since the waste may neverbe removed, waste could be collected in rigid, semi-rigid, or flexiblechambers.

It is within the scope of this invention to do the second-stageamplification with the same primers used in the first-stageamplification (see U.S. Pat. No. 6,605,451). However, it is oftenadvantageous to have primers in second-stage reactions that are internalto the first-stage product such that there is no or minimal overlapbetween the first- and second-stage primer binding sites. Dilution offirst-stage product largely eliminates contribution of the originaltemplate DNA and first-stage reagents to the second-stage reaction.Furthermore, illustratively, second-stage primers with a Tm higher thanthose used in the first-stage may be used to potentiate nestedamplification. Illustratively, second-stage products may be betweenabout 100 to about 140 base pairs and have Tm values of 65° C.+/−2° C.Tm of about 65° C. allows effective two-temperature amplification. Insecond-stage amplification, illustrative parameters of 94° C. for 0-5seconds transitioning to 65° C. for 15 seconds are anticipated. Primermay be designed to avoid significant hairpins, hetero/homo-dimers andundesired hybridization. Because of the nested format, second-stageprimers tolerate deleterious interactions far more so than primers usedto amplify targets from genomic DNA in a single step. Optionally,hot-start is used on second-stage amplification.

If a fluorescent dye is included in second-stage amplification,illustratively as a dsDNA binding dye or as part of a fluorescent probe,as are known in the art, optics provided may be used to monitoramplification of one or more of the samples. Optionally, analysis of theshape of the amplification curve may be provided to call each samplepositive or negative. Illustrative methods of calling the sample arediscussed in U.S. Pat. No. 6,730,501, herein incorporated by reference.Alternatively, methods employing a crossing threshold may be used. Acomputer may be provided externally or within the instrument and may beconfigured to perform the methods and call the sample positive ornegative based upon the presence or absence of second-stageamplification. It is understood, however, that other methods, as areknown in the art, may be used to call each sample. Other analyses may beperformed on the fluorescent information. One such non-limiting exampleis the use of melting curve analysis to show proper meltingcharacteristics (e.g. Tm, melt profile shape) of the amplicon. Theoptics provided may be configured to capture images of all blisters 82at once, or individual optics may be provided for each individualblister. Other configurations are within the scope of this invention.

FIG. 5 shows an alternative pouch 110. In this embodiment, variousreagents are loaded into pouch 110 via fitment 190. FIG. 5 a shows across-section of fitment 190 with one of a plurality of plungers 168. Itis understood that, while FIG. 5 a shows a cross-section through entrychannel 115 a, as shown in the embodiment of FIG. 5 , there are 12 entrychannels present (entry channel 115 a through 115 l), each of which mayhave its own plunger 168 for use in fitment 190, although in thisparticular configuration, entry channels 115 c, 115 f, and 115 i are notused. It is understood that a configuration having 12 entry channels isillustrative only, and that any number of entry channels and associatedplungers may be used. In the illustrative embodiment, an optional vacuumport 142 of fitment 190 is formed through a first surface 194 of fitment190 to communicate with chamber 192. Optional vacuum port 142 may beprovided for communication with a vacuum or vacuum chamber (not shown)to draw out the air from within pouch 110 to create a vacuum withinchamber 192 and the various blisters and chambers of pouch 110. Plunger168 is then inserted far enough into chamber 192 to seal off vacuum port142. Chamber 192 is illustratively provided under a predetermined amountof vacuum to draw a desired volume of liquid into chamber 192 upon use.Additional information on preparing chamber 192 may be found in U.S.patent application Ser. No. 10/512,255, already incorporated byreference.

Illustrative fitment 190 further includes an injection port 141 formedin the second surface 195 of fitment 190. Illustratively, injection port141 is positioned closer to the plastic film portion of pouch 110 thanvacuum port 142, as shown in FIG. 5 a , such that the plunger 168 isinserted far enough to seal off vacuum port 142, while still allowingaccess to chamber 192 via injection port 141. As shown, second surface119 of plastic film portion 117 provides a penetrable seal 139 toprevent communication between chamber 192 and the surrounding atmospherevia injection port 141. However, it is understood that second surface119 optionally may not extend to injection port 141 and various otherseals may be employed. Further, if another location for the seal isdesired, for example on a first surface 194 of fitment 190, injectionport 141 may include a channel to that location on fitment 190. U.S.patent application Ser. No. 10/512,255, already incorporated byreference, shows various configurations where the seal is locatedremotely from the injection port, and the seal is connected to thechamber via a channel. Also, U.S. patent application Ser. No. 10/512,255discloses various configurations where channels connect a single seal tomultiple chambers. Variations in seal location, as well as connection ofa single injection port to multiple chambers, are within the scope ofthis invention. Optionally, seal 139 may be frangible and may be brokenupon insertion of a cannula (not shown), to allow a fluid sample fromwithin the cannula to be drawn into or forced into chamber 192.

The illustrative plunger 168 of the pouch assembly 110 is cylindrical inshape and has a diameter of approximately 5 mm to be press-fit intochamber 192. Plunger 168 includes a first end portion 173 and anopposite second end portion 175. As shown in FIG. 5 a , a notch 169 ofplunger 168 is formed in second end portion 175. In use, second endportion 175 is inserted part way into chamber 192, and notch 169 may bealigned with injection port 141 to allow a fluid sample to be drawn intoor injected into chamber 192, even when plunger 168 is inserted farenough that plunger 168 would otherwise be blocking injection port 141.

Illustratively, a fluid is placed in a container (not shown) with asyringe having a cannulated tip that can be inserted into injection port141 to puncture seal 139 therein. In using an air-evacuated pouchassembly 110, when seal 139 is punctured, the fluid is withdrawn fromthe container due to the negative pressure within chamber 192 relativeto ambient air pressure. Fluid then passes through port 141 to fillchamber 192. At this point, the fluid usually does not flow into theplastic film portion 117 of pouch 110. Finally, the plunger 168 isinserted into chamber 192 such that second end portion 175 of plunger168 approaches the bottom 191 of chamber 192, to push a measured amountof the reagent or sample into the plastic film portion 117. As shown,plunger 168 is configured such that upon full insertion, second endportion 175 does not quite meet bottom 191 of chamber 192. The remainingspace is useful in trapping bubbles, thereby reducing the number ofbubbles entering plastic film portion 117. However, in some embodimentsit may be desirable for second end portion 175 to meet bottom 191 uponfull insertion of plunger 168. In the embodiment shown in FIG. 5 , entrychannels 115 a, 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and 115l all lead to reaction zones or reservoir blisters. It is understoodthat full insertion of the plunger associated with entry channel 115 awould force a sample into three-lobed blister 122, full insertion of theplunger associated with entry channel 115 b would force a reagent intoreservoir blister 101, full insertion of the plunger associated withentry channel 115 d would force a reagent into reservoir blister 102,full insertion of the plunger associated with entry channel 115 e wouldforce a reagent into reservoir blister 103, full insertion of theplunger associated with entry channel 115 g would force a reagent intoreservoir blister 104, full insertion of the plunger associated withentry channel 115 h would force a reagent into reservoir blister 105,full insertion of the plunger associated with entry channel 115 j wouldforce a reagent into reservoir blister 106, full insertion of theplunger associated with entry channel 115 k would force a reagent intoreservoir blister 107, and full insertion of the plunger associated withentry channel 115 l would force a reagent into reservoir blister 108.

If a plunger design is used including notch 169 as illustrated in theembodiment shown in FIG. 5 a , the plunger 168 may be rotated prior tobeing lowered, so as to offset notch 169 and to close off injection port141 from communication with chamber 192, to seal the contents therein.This acts to minimize any potential backflow of fluid through injectionport 141 to the surrounding atmosphere, which is particularly usefulwhen it is desired to delay in full insertion of the plunger. Althoughnotch 169 is shown and described above with respect to plunger 168, itis within the scope of this disclosure to close off injection port 141soon after dispensing the fluid sample into the chamber 192 by othermeans, such as depressing plunger 168 toward the bottom of chamber 192,heat sealing, unidirectional valves, or self-sealing ports, for example.If heat sealing is used as the sealing method, a seal bar could beincluded in the instrument such that all chambers are heat sealed uponinsertion of the pouch into the instrument.

In the illustrative method, the user injects the sample into theinjection port 141 associated with entry channel 115 a, and water intothe various other injection ports. The water rehydrates reagents thathave been previously freeze-dried into chambers 192 associated with eachof entry channels 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and115 l. The water may be injected through one single seal and then bedistributed via a channel to each of the chambers, as shown in FIG. 6below, or the water could be injected into each chamber independently.Alternatively, rather than injecting water to rehydrate dried reagents,wet reagents such as lysis reagents, nucleic acid extraction reagents,and PCR reagents may be injected into the appropriate chambers 192 ofthe fitment 190.

Upon activation of the plunger 168 associated with entry channel 115 a,the sample is forced directly into three-lobed blister 122 via channel114. The user also presses the remaining plungers 168, forcing thecontents out of each of the chambers 192 in fitment 190 and intoreservoir blisters 101 through 108. At this point, pouch 110 is loadedinto an instrument for processing. While instrument 800, shown in FIG. 8, is configured for the pouch 210 of FIG. 6 , it is understood thatmodification of the configuration of the bladders of instrument 800would render instrument 800 suitable for use with pouch 110, or withpouches of other configurations.

In one illustrative example, upon depression of the plungers 168,reservoir blister 101 now contains DNA-binding magnetic beads inisopropanol, reservoir blister 102 now contains a first wash solution,reservoir blister 103 now contains a second wash solution, reservoirblister 104 now contains a nucleic acid elution buffer, reservoirblister 105 now contains first-stage PCR reagents, including multiplexedfirst-stage primers, reservoir blister 106 now contains second-stage PCRreagents without primers, reservoir blister 107 now contains negativecontrol PCR reagents without primers and without template, and reservoirblister 108 now contains positive control PCR reagents with template.However, it is understood that these reagents are illustrative only, andthat other reagents may be used, depending upon the desired reactionsand optimization conditions.

Once pouch 110 has been placed into instrument 800 and the sample hasbeen moved to three-lobed blister 122, the sample may be subjected todisruption by agitating the sample with lysing particles such as ZS orceramic beads. The lysing particles may be provided in three-lobedblister 122, or may be injected into three-lobed blister 122 along withthe sample. The three-lobed blister 122 of FIG. 5 is operated in muchthe same way as three-lobed blister 22 of FIG. 1 , with the two lowerlobes 124, 126 pressurized together, and pressure is alternated betweenthe upper lobe 128 and the two lower lobes 124, 126. However, asillustrated, lower lobes 124, 126 are much more rounded than lower lobes24, 26, allowing for a smooth flow of beads to channel 130 and allowingfor high-speed collisions, even without the triangular flow separator atnexus 32. As with three-lobed blister 22, three-lobed blister 122 ofFIG. 5 allows for effective lysis or disruption of microorganisms,cells, and viral particles in the sample. It has been found that achannel 130 having a width of about 3-4 mm, and illustratively about 3.5mm, remains relatively clear of beads during lysis and is effective inproviding for high-velocity collisions.

After lysis, nucleic-acid-binding magnetic beads are injected into upperlobe 128 via channel 138 by pressurizing a bladder positioned overreservoir blister 101. The magnetic beads are mixed, illustratively moregently than with during lysis, with the contents of three-lobed blister122, and the solution is incubated, illustratively for about 1 minute,to allow nucleic acids to bind to the beads.

The solution is then pumped into the “FIG. 8 ” blister 144 via channel143, where the beads are captured by a retractable magnet housed in theinstrument, which is illustratively pneumatically driven. The beadcapture process begins by pressurizing all lobes 124, 126, and 128 ofthe bead milling apparatus 122. This forces much of the liquid contentsof 122 through channel 143 and into blister 144. A magnet is broughtinto contact with the lower portion 144 b of blister 144 and the sampleis incubated for several seconds to allow the magnet to capture thebeads from the solution, then the bladders adjacent to blister 122 aredepressurized, the bladders adjacent blister portions 144 a and 144 bare pressurized, and the liquid is forced back into blister 122. Sincenot all of the beads are captured in a single pass, this process may berepeated up to 10 times to capture substantially all of the beads inblister 144. Then the liquid is forced out of blister 144, leavingbehind only the magnetic beads and the captured nucleic acids, and washreagents are introduced into blister 144 in two successive washes (fromreservoir blisters 102 and 103 via channels 145 and 147, respectively).In each wash, the bladder positioned over the reservoir blistercontaining the wash reagent is pressurized, forcing the contents intoblister 144. The magnet is withdrawn and the pellet containing themagnetic beads is disrupted by alternatively pressurizing each of twobladders covering each lobe 144 a and 144 b of blister 144. When theupper lobe 144 a is compressed, the liquid contents are forced into thelower lobe 144 b, and when the lower lobe 144 b is compressed, thecontents are forced into the upper lobe 144 a. By agitating the solutionin blister 144 between upper lobe 144 a and lower lobe 144 b, themagnetic beads are effectively washed of impurities. A balance ismaintained between inadequate agitation, leaving the pellet of beadsundisturbed, and excessive agitation, potentially washing the nucleicacids from the surface of the beads and losing them with the washreagents. After each wash cycle, the magnetic beads are captured via themagnet in blister 144 and the wash reagents are illustratively forcedinto three-lobed blister 122, which now serves as a waste receptacle.However, it is understood that the used reservoir blisters may alsoserve as waste receptacles, or other blisters may be providedspecifically as waste receptacles.

Nucleic acid elution buffer from reservoir blister 104 is then injectedvia channel 149 into blister 144, the sample is once again agitated, andthe magnetic beads are recaptured by employment of the magnet. The fluidmixture in blister 144 now contains nucleic acids from the originalsample. Pressure on blister 144 moves the nucleic acid sample to thefirst stage PCR blister 161 via channel 152, where the sample is mixedwith first-stage PCR master mix containing multiple primer sets, the PCRmaster mix provided from reservoir blister 105 via channel 162. Ifdesired, the sample and/or the first-stage PCR master mix may be heatedprior to mixing, to provide advantages of hot start. As will be seenbelow, pouch 110 of FIG. 5 is configured for up to 10 primer sets, butit is understood that the configuration may be altered and any number ofprimer sets may be used. A bladder positioned over blister 161 ispressurized at low pressure, to force the contents of blister 161 intointimate contact with a heating/cooling element, illustratively aPeltier element, on the other side of blister 161. The pressure onblister 161 should be sufficient to assure good contact with theheating/cooling element, but should be gentle enough such that fluid isnot forced from blister 161. The heating/cooling element is temperaturecycled, illustratively between about 60° C. to about 95° C.Illustratively, temperature cycling is performed for about 15-20 cycles,resulting in amplification of one or more nucleic acid targets present.Also illustratively, temperature cycling ceases prior to plateau phase,and may cease in log phase or even prior to log phase. In one example,it may be desirable merely to enrich the sample with the desiredamplicons, without reaching minimal levels of detection. See U.S. Pat.No. 6,605,451, herein incorporated by reference.

The amplified sample is optionally then diluted by forcing most thesample back into blister 144 via channel 152, leaving only a smallamount (illustratively about 1 to 5%) of the amplified sample in blister161, and second-stage PCR master mix is provided from reservoir blister106 via channel 163. The sample is thoroughly mixed illustratively bymoving it back and forth between blisters 106 and 161 via channel 163.If desired, the reaction mixture may be heated prior to second-stageamplification. The sample is then forced through channel 165 into anarray of low volume blisters 182 in the center of second-stageamplification zone 180. Each of the ten illustrative low volume blisters182 may contain a different primer pair, either essentially the same asone of the primer pairs in the first-stage amplification, or “nested”within the first-stage primer pair to amplify a shortened amplicon. Theprimers, now hydrated by the sample, complete the amplification mixture.Positive and negative control samples are also introduced bypressurizing the contents of reservoir blisters 107 and 108,respectively, forcing PCR master mix either without target DNA fromreservoir blister 107 via channel 166, or with control DNA fromreservoir blister 108, via channel 167. As illustrated, there are fiveeach of positive control blisters 183 and negative control blisters 181,which may be multiplexed 2-fold to provide the necessary controls forten different second-stage amplification reactions. It is understoodthat this configuration is illustrative only and that any number ofsecond-stage blisters may be provided.

Illustratively, the PCR master mix used for second-stage amplificationlacks the primers, but is otherwise complete. However, an “incomplete”PCR master mix may lack other PCR components as well. In one example,the second-stage PCR master mix is water or buffer only, which is thenmixed with the optionally diluted first-stage PCR amplification product.This mixture is moved to the small-volume PCR reaction blisters, whereall of the remaining components have been previously provided. Ifdesired, all of the remaining components may be mixed together andspotted as a single mixture into the small-volume PCR reaction blisters.Alternatively, as illustrated in FIG. 5 b , each of the components maybe spotted onto a separate region of the small-volume PCR reactionblister 182. As shown in FIG. 5 b , four regions are present,illustratively with dNTPs spotted at region 182 a, primers spotted at,polymerase spotted at 182 c, and a magnesium compound spotted at 182 d.By spotting the components separately and heating the sample mixtureprior to rehydrating the components, nonspecific reactions can beminimized. It is understood that any combination of components can bespotted this way, and that this method of spotting components into oneor more regions of the blisters may be used with any embodiment of thepresent invention.

The channels 165, 166, and 167 leading to the small-volume PCR reactionblisters 181, 182, and 183 are sealed, and a pneumatic bladder gentlypresses the array against a heating/cooling element, illustratively aPeltier element, for thermal cycling. The cycling parameters may beindependently set for second-stage thermal cycling. Illustratively, thereactions are monitored by focusing an excitation source, illustrativelya blue light (450-490 nm), onto the array, and imaging the resultantfluorescent emissions, illustratively fluorescent emissions above 510nm.

In the above example, pinch valves are not discussed. However, it isunderstood that when it is desired to contain a sample in one of theblisters, pneumatic actuators positioned over channels leading to andfrom the particular blister are pressurized, creating pinch valves andclosing off the channels. Conversely, when it is desired to move asample from one of the blisters, the appropriate pneumatic actuator isdepressurized, allowing the sample flow through the channel.

The pouch described above in FIG. 5 includes reagent reservoir blisters101 through 108, in which the user injected reagents from the fitment190 into the reagent reservoir blisters 101 through 108 in the plasticfilm portion 117 of the pouch 110, illustratively prior to insertion ofpouch 110 into the instrument. While there are advantages to the use ofthe reagent reservoir blisters of FIG. 5 , including having the abilityto maintain the contents of the various blisters at differenttemperatures, there are some disadvantages as well. Because the operatoris responsible for moving the reagents from the fitment 190 to thereservoir blisters 101 through 108, and because this is often doneoutside of the machine and thus without activated pinch valves, reagentscould occasionally leak from the reservoir blisters to the workingblisters. The reagents in reservoir blisters are exposed duringpreparation and loading. If they are pressed, squeezed, or even lightlybumped, the reagents may leak through available channels. If the loss ofreagents is substantial, the reaction may fail completely. Furthermore,during operation there may be some variability in the amount of reagentforced from the reservoir blisters 101 through 108, leading toinconsistent results. Automation of introduction of the reagents tofitment 190 and movement of the reagents from fitment 190 to reagentreservoir blisters 101 through 108 would solve many of these problems,and is within the scope of this invention.

The pouch 210 of FIG. 6 addresses many of these issues in a differentway, by using a direct-injection approach wherein the instrument itselfmoves the plungers 268, illustratively via pneumatic pistons, and forcesthe reagents into the various working blisters as the reagents areneeded. Rather than storing the reagents in reservoir blisters 101through 108 of FIG. 5 , in the embodiment of FIG. 6 the reagents areintroduced into various chambers 292 of fitment 290 and are maintainedthere until needed. Pneumatic operation of piston 268 at the appropriatetime introduces a measured amount of the reagent to the appropriatereaction blister. In addition to addressing many of the above-mentionedissues, pouch 210 also has a much more compact shape, allowing for asmaller instrument design, and pouch 210 has shorter channels,permitting better fluid flow and minimizing reagent loss in channels.

In one illustrative embodiment of FIG. 6 , a 300 μl mixture comprisingthe sample to be tested (100 μl) and lysis buffer (200 μl) is injectedinto injection port 241 a. Water is also injected into the fitment 290via seal 239 b, hydrating up to eleven different reagents, each of whichwere previously provided in dry form in chambers 292 b through 292 l viachannel 293 (shown in shadow). These reagents illustratively may includefreeze-dried PCR reagents, DNA extraction reagents, wash solutions,immunoassay reagents, or other chemical entities. For the example ofFIG. 6 , the reagents are for nucleic acid extraction, first-stagemultiplex PCR, dilution of the multiplex reaction, and preparation ofsecond-stage PCR reagents, and control reactions. In the embodimentshown in FIG. 6 , all that need be injected is the sample in port 241 aand water in port 241 b.

As shown in FIG. 6 , water injected via seal 293 b is distributed tovarious chambers via channel 293. In this embodiment, only the sampleand water need be injected into pouch 210. It is understood, however,that water could be injected into each chamber 292 independently.Further, it is understood that, rather than providing dried reagents inthe various chambers 292 and hydrating upon injection of the water,specific wet reagents could be injected into each chamber, as desired.Additionally, it is understood that one or more of chambers 292 could beprovided with water only, and the necessary reagents may be provideddried in the appropriate reaction blisters. Various combinations of theabove, as dictated by the needs of the particular reaction, are withinthe scope of this invention.

As seen in FIG. 6 , optional protrusions 213 are provided on bottomsurface 297 of fitment 290. As shown, protrusions 213 are located withintheir respective entry channels 215. However, other configurations arepossible. Protrusions 213 assist in opening entry channel 215 andprevent bottom surface 297 from engaging another flat surface in such away to pinch off entry channels 215 when plungers 268 are depressed,which helps prevent back-flow upon activation of the plungers 268. Suchprotrusions may be used on any of the various pouches according to thepresent invention.

In embodiments wherein water is injected into the pouch to hydratemultiple dry reagents in multiple chambers in the fitment, a means ofclosing the channel between the injection port and the many chambers isdesired. If the channel is not closed, activation of each plunger mayforce some of the contents of its respective chamber back out into thechannel, potentially contaminating neighboring chambers and altering thevolumes contained in and delivered from the chamber. Several ways ofclosing this channel have been used, including rotating a notchedplunger 268 as discussed above, and heat-sealing the plastic film acrossthe channel thereby closing the channel permanently, and applyingpressure to the channel as a pinch valve. Other closures may also beused, such as valves built into the fitment, illustratively one-wayvalves.

After the fluids are loaded into chambers 292 and pouch 210 is loadedinto the instrument, plunger 268 a is depressed illustratively viaactivation of a pneumatic piston, forcing the balance of the sample intothree-lobed blister 220 via channel 214. As with the embodiments shownin FIGS. 1 and 5 , the lobes 224, 226, and 228 of three-lobed blister220 are sequentially compressed via action bladders 824, 826, and 828 ofbladder assembly 810, shown in FIGS. 7-9 , forcing the liquid throughthe narrow nexus 232 between the lobes, and driving high velocitycollisions, shearing the sample and liberating nucleic acids,illustratively including nucleic acids from hard-to-open spores,bacteria, and fungi. Cell lysis continues for an appropriate length oftime, illustratively 0.5 to 10 minutes.

Once the cells have been adequately lysed, plunger 268 b is activatedand nucleic acid binding magnetic beads stored in chamber 292 b areinjected via channel 236 into upper lobe 228 of three-lobed blister 220.The sample is mixed with the magnetic beads and the mixture is allowedto incubate for an appropriate length of time, illustrativelyapproximately 10 seconds to 10 minutes.

The mixture of sample and beads are forced through channel 238 intoblister 244 via action of bladder 826, then through channel 243 and intoblister 246 via action of bladder 844, where a retractable magnet 850located in instrument 800 adjacent blister 245, shown in FIG. 8 ,captures the magnetic beads from the solution, forming a pellet againstthe interior surface of blister 246. A pneumatic bladder 846, positionedover blister 246 then forces the liquid out of blister 246 and backthrough blister 244 and into blister 222, which is now used as a wastereceptacle. However, as discussed above with respect to FIG. 5 , otherwaste receptacles are within the scope of this invention. One ofplungers 268 c, 268 d, and 268 e may be activated to provide a washsolution to blister 244 via channel 245, and then to blister 246 viachannel 243. Optionally, the magnet 850 is retracted and the magneticbeads are washed by moving the beads back and forth from blisters 244and 246 via channel 243, by alternatively pressurizing bladders 844 and846. Once the magnetic beads are washed, the magnetic beads arerecaptured in blister 246 by activation of magnet 850, and the washsolution is then moved to blister 222. This process may be repeated asnecessary to wash the lysis buffer and sample debris from the nucleicacid-binding magnetic beads. Illustratively, three washes are done, oneeach using wash reagents in chambers 292 c, 292 d, and 292 e. However,it is understood that more or fewer washes are within the scope of thisinvention. If more washes are desired, more chambers 292 may beprovided. Alternatively, each chamber 292 may hold a larger volume offluid and activation of the plungers may force only a fraction of thevolume from the chamber upon each activation.

After washing, elution buffer stored in chamber 292 f is moved viachannel 247 to blister 248, and the magnet is retracted. The solution iscycled between blisters 246 and 248 via channel 252, breaking up thepellet of magnetic beads in blister 246 and allowing the capturednucleic acids to dissociate from the beads and come into solution. Themagnet 850 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is forced into blister248.

Plunger 268 h is depressed and first-stage PCR master mix from chamber292 h is mixed with the nucleic acid sample in blister 248. Optionally,the mixture is mixed by alternative activation of bladders 848 and 864,forcing the mixture between 248 and 264 via channel 253. After severalcycles of mixing, the solution is contained in blister 264, wherefirst-stage multiplex PCR is performed. If desired, prior to mixing, thesample may be retained in blister 246 while the first-stage PCR mastermix is pre-heated, illustratively by moving the first-stage PCR mastermix into blister 264 or by providing a heater adjacent blister 248. Asdiscussed above, this pre-heating may provide the benefits of hot startPCR. The instrument 800 illustrated in FIG. 8 features Peltier-basedthermal cyclers 886 and 888 which heat and cool the sample. However, itis understood that other heater/cooler devices may be used, as discussedabove. Temperature cycling is illustratively performed for 15-20 cycles,although other levels of amplification may be desirable, depending onthe application, as discussed above. As will be seen below, thesecond-stage amplification zone 280 is configured to detectamplification in 18 second-stage reactions. Accordingly, 18 differentprimer-pairs may be included in the PCR reaction in blister 264.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted as discussed above with respect to theembodiment of FIG. 5 , by forcing most of the sample back into blister248, leaving only a small amount, and adding second-stage PCR master mixfrom chamber 292 i. Alternatively, a dilution buffer from 292 i may bemoved to blister 266 via channel 249 and then mixed with the amplifiedsample in blister 264 by moving the fluids back and forth betweenblisters 264 and 266. After mixing, a portion of the diluted sampleremaining in blister 264 is forced away to three-lobed blister 222, nowthe waste receptacle. If desired, dilution may be repeated severaltimes, using dilution buffer from chambers 292 j and 292 k, and thenadding second-stage PCR master mix from chamber 292 g to some or all ofthe diluted amplified sample. It is understood that the level ofdilution may be adjusted by altering the number of dilution steps or byaltering the percentage of the sample discarded prior to mixing with thedilution buffer or second-stage PCR master mix. If desired, this mixtureof the sample and second-stage PCR master mix may be pre-heated inblister 264 prior to movement to second-stage blisters 282 forsecond-stage amplification.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 18 second-stage blisters 282 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may then bepre-loaded in the second-stage blisters 282 as well. As discussed abovewith the prior examples, each primer pair may be identical to afirst-stage PCR primer pair or may be nested within the first-stageprimer pair. Movement of the sample from blister 264 to the second-stageblisters completes the PCR reaction mixture. Control samples fromchamber 292 l are also moved to control blisters 283 via channel 267.The control samples may be positive or negative controls, as desired.Illustratively, each pouch would contain control reactions that validatethe operation of each step in the process and demonstrate that positiveresults are not the result of self-contamination with previouslyamplified nucleic acids. However, this is not practical in manyprotocols, particularly for a highly multiplexed reaction. Oneillustrative way of providing suitable controls involves spiking sampleswith a species such as baker's yeast. The nucleic acids are extractedfrom the yeast, alongside other nucleic acids. First- and second-stagePCR reactions amplify DNA and/or RNA targets from the yeast genome.Illustratively, an mRNA sequence derived from a spliced pre-mRNA can beused to generate an RNA-specific target sequence by arranging the primersequences to span an intron. A quantitative analysis of the yeast copynumber against reference standards allows substantial validation thateach component of the system is working. Negative control reactions foreach of the many second-stage assays are more problematic. It may bedesirable to run control reactions either in parallel or in a separaterun.

Activation of bladder 882 of bladder assembly 810 seals the samples intotheir respective second-stage blisters 282, 283, and activation ofbladder 880 provides gentle pressure on second-stage blisters 282, 283,to force second-stage blisters 282, 283 into contact with aheater/cooler device. A window 897 positioned over the second-stageamplification zone 280 allows fluorescence monitoring of the arrayduring PCR and during a DNA melting-curve analysis of the reactionproducts.

It is noted that the pouch 210 of FIG. 6 has several unsealed areas,such as unsealed area 255 and unsealed area 256. These unsealed areasform blisters that are not involved in any of the reactions in thisillustrative embodiment. Rather, these unsealed areas are provided inspace between the working blisters and channels. As compared to pouchesthat are sealed in all unused space, it has been found that fewer leaksresult when unsealed areas such as 255 and 256 are provided, presumablyby reducing problematic wrinkles in the film material. Such unsealedareas may be provided on any pouch embodiment.

FIG. 8 shows an illustrative apparatus 800 that could be used with pouch210. Instrument 800 includes a support member 802 that could form a wallof a casing or be mounted within a casing. Instrument 800 also includesa second support member 804 that is movable with respect to supportmember 802, to allow insertion and withdrawal of pouch 210. Movablesupport member 804 may be mounted on a track or may be moved relative tosupport member 802 in any of a variety of ways. Illustratively, a lid805 fits over pouch 210 once pouch 210 has been inserted into instrument800.

Illustratively, the bladder assembly 810 and pneumatic valve assembly808 are mounted on movable member 802, while the heaters 886 and 888 aremounted on support member 802. However, it is understood that thisarrangement is illustrative only and that other arrangements arepossible. As bladder assembly 810 and pneumatic valve assembly 808 aremounted on movable support member 804, these pneumatic actuators may bemoved toward pouch 210, such that the pneumatic actuators are placed incontact with pouch 210. When pouch 210 is inserted into instrument 800and movable support member 804 is moved toward support member 802, thevarious blisters of pouch 210 are in a position adjacent to the variouspneumatic bladders of bladder assembly 810 and the various pneumaticpistons of pneumatic valve assembly 808, such that activation of thepneumatic actuators may force liquid from one or more of the blisters ofpouch 210 or may form pinch valves with one or more channels of pouch210. The relationship between the blisters and channels of pouch 210 andthe pneumatic actuators of bladder assembly 810 and pneumatic valveassembly 808 are discussed in more detail below with respect to FIGS. 9and 10 .

Each pneumatic actuator has one or more pneumatic fittings. For example,bladder 824 of bladder assembly 810 has pneumatic fitting 824 a andpneumatic piston 843 has its associated pneumatic fitting 843 a. In theillustrative embodiment, each of the pneumatic fittings of bladderassembly 810 extends through a passageway 816 in movable support member804, where a hose 878 connects each pneumatic fitting to compressed airsource 895 via valves 899. In the illustrative embodiment, thepassageways 816 not only provide access to compressed air source 895,but the passageways also aid in aligning the various components ofbladder assembly 810, so that the bladders align properly with theblisters of pouch 210.

Similarly, pneumatic valve assembly 808 is also mounted on movablesupport member 804, although it is understood that other configurationsare possible. In the illustrative embodiment, pins 858 on pneumaticvalve assembly 808 mount in mounting openings 859 on movable supportmember 804, and pneumatic pistons 843, 852, 853, and 862 extend throughpassageways 816 in movable support member 804, to contact pouch 210. Asillustrated, bladder assembly is mounted on a first side 811 of movablesupport member 804 while pneumatic valve assembly 808 is mounted on asecond side 812 of movable support member 804. However, becausepneumatic pistons 843, 852, 853, and 862 extend through passageways 816,the pneumatic pistons of pneumatic valve assembly 808 and the pneumaticbladders of bladder assembly 810 work together to provide the necessarypneumatic actuators for pouch 210.

As discussed above, each of the pneumatic actuators of bladder assembly810 and pneumatic valve assembly 808 has an associated pneumaticfitting. While only several hoses 878 are shown in FIG. 8 , it isunderstood that each pneumatic fitting is connected via a hose 878 tothe compressed gas source 895. Compressed gas source 895 may be acompressor, or, alternatively, compressed gas source 895 may be acompressed gas cylinder, such as a carbon dioxide cylinder. Compressedgas cylinders are particularly useful if portability is desired. Othersources of compressed gas are within the scope of this invention.

Several other components of instrument 810 are also connected tocompressed gas source 895. Magnet 850, which is mounted on a first side813 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 246 of pouch 210. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 246, and when magnet 850 is retracted, magnet 850 doesnot significantly affect any magnetic beads present in blister 246.While reference is made to retracting magnet 850, it is understood thatan electromagnet may be used and the electromagnet may be activated andinactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field.

The various pneumatic pistons 868 of pneumatic piston array 869, whichis mounted on support 802, are also connected to compressed gas source895 via hoses 878. While only two hoses 878 are shown connectingpneumatic pistons 868 to compressed gas source 895, it is understoodthat each of the pneumatic pistons 868 are connected to compressed gassource 895. Twelve pneumatic pistons 868 are shown. When the pouch 210is inserted into instrument 800, the twelve pneumatic pistons 868 arepositioned to activate their respective twelve plungers 268 of pouch210. When lid 805 is closed over pouch 210, a lip 806 on lid 805provides a support for fitment 290, so that as the pneumatic pistons 868are activated, lid 805 holds fitment 290 in place. It is understood thatother supports for fitment 290 are within the scope of this invention.

A pair of heating/cooling devices, illustratively Peltier heaters, aremounted on a second side 814 of support 802. First-stage heater 886 ispositioned to heat and cool the contents of blister 264 for first-stagePCR. Second-stage heater 888 is positioned to heat and cool the contentsof second-stage blisters 282 and 283 of pouch 210, for second-stage PCR.It is understood, however, that these heaters could also be used forother heating purposes, and that other heaters may be included, asappropriate for the particular application.

If desired, a feedback mechanism (not shown) may be included ininstrument 800 for providing feedback regarding whether the sample hasactually been forced into a particular blister. Illustrative feedbackmechanisms include temperature or pressure sensors or optical detectors,particularly if a fluorescent or colored dye is included. Such feedbackmechanisms illustratively may be mounted on either of support members802 or 804. For example, a pressure sensor may be mounted on support 802adjacent the location of blister 264. When the sample is supposedlymoved to blister 264, if the pressure sensor is depressed, then sampleprocessing is allowed to continue. However, if the pressure sensor isnot depressed, then sample processing may be stopped, or an errormessage may be displayed on screen 892. Any combination or all of theblisters may have feedback mechanisms to provide feedback regardingproper movement of the sample through the pouch.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 8 , optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. A window 897 through movablesupport 804 provides optical array 890 with access to second-stageamplification zone 280 of pouch 210. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage blister282, 823 in pouch 210. Alternatively, camera 896 may take images thatcontain all of the second-stage blisters 282, 283, and the image may bedivided into separate fields corresponding to each of the second-stageblisters 282, 283. Depending on the configuration, optical array 890 maybe stationary, or optical array 890 may be placed on movers attached toone or more motors and moved to obtain signals from each individualsecond-stage blister 282, 283. It is understood that other arrangementsare possible.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Computer894 also controls heaters 886 and 888, and optical array 890. Each ofthese components is connected electrically, illustratively via cables891, although other physical or wireless connections are within thescope of this invention. It is understood that computer 894 may behoused within instrument 890 or may be external to instrument 890.Further, computer 894 may include built-in circuit boards that controlsome or all of the components, and may also include an externalcomputer, such as a desktop or laptop PC, to receive and display datafrom the optical array. An interface 893, illustratively a keyboardinterface, may be provided including keys for inputting information andvariables such as temperatures, cycle times, etc. Illustratively, adisplay 892 is also provided. Display 892 may be an LED, LCD, or othersuch display, for example.

FIG. 9 shows the relationship between bladder assembly 810 and pouch 210during operation of instrument 800. Bladder assembly comprisessub-assemblies 815, 817, 818, 819, and 822. Because bladder 809 ofbladder sub-assembly 815 is large, bladder sub-assembly 815illustratively has two pneumatic fittings 815 a and 815 b. Bladder 809is used to close off chambers 292 (as shown in FIG. 6 ) from the plasticfilm portion 217 of pouch 210. When one of the plungers 268 isdepressed, one or both of pneumatic fittings 815 a and 815 b permitbladder 809 to deflate. After the fluid from one of the chambers 292passes through, bladder 809 is re-pressurized, sealing off channels 214,236, 245, 247, and 249. While illustrative bladder sub-assembly 815 hasonly one bladder 809, it is understood that other configurations arepossible, illustratively where each of channels 214, 236, 245, 247, and249 has its own associated bladder or pneumatic piston. Bladdersub-assembly 822 illustratively comprises three bladders 824, 826, and828. As discussed above, bladders 824, 824, and 828 drive thethree-lobed blister 222 for cell lysis. As illustrated, bladders 824,826, and 828 are slightly larger than their corresponding blisters 224,226, 228. It has been found that, upon inflation, the surface of thebladders can become somewhat dome-shaped, and using slightly oversizedbladders allows for good contact over the entire surface of thecorresponding blister, enabling more uniform pressure and betterevacuation of the blister. Bladder sub-assembly 817 has four bladders.Bladder 836 functions as a pinch-valve for channel 236, while bladders844, 848, and 866 are configured to provide pressure on blisters 244,248, and 266, respectively. Bladder sub-assembly 818 has two bladders846 and 864, which are configured to provide pressure on blisters 246and 264, respectively. Finally, bladder sub-assembly 819 controlssecond-stage amplification zone 280. Bladder 865 acts as a pinch valvefor channels 265 and 267, while bladder 882 provides gentle pressure tosecond-stage blisters 282 and 283, to force second-stage blisters intoclose contact with heater 888. While bladder assembly 810 is providedwith five sub-assemblies, it is understood that this configuration isillustrative only and that any number of sub-assemblies could be used orthat bladder assembly 810 could be provided as a single integralassembly.

FIG. 10 similarly shows the relationship between pneumatic valveassembly 808 and pouch 210 during operation of instrument 800. Ratherthan bladders, pneumatic valve assembly 808 has four pneumatic pistons842, 852, 853, and 862. These pneumatic pistons 842, 852, 853, and 862,each driven by compressed air, provide directed pressure on channels242, 252, 253, and 262. Because the pistons are fairly narrow indiameter, they can fit between bladder sub-assembly 817 and bladdersub-assembly 818 to provide pinch valves for channels 242, 252, 253, and262, allowing channels 242, 252, 253, and 262 to be fairly short.However, if desired, pneumatic pistons 842, 852, 853, and 862 could bereplaced by bladders, which may be included in bladder assembly 810,obviating the need for pneumatic valve assembly 808. It is understoodthat any combination of bladders and pneumatic pistons are within thescope of this invention. It is also understood that other methods ofproviding pressure on the channels and blisters of pouch 210, as areknown in the art, are within the scope of this invention.

Example 1: Nested Multiplex PCR

A set of reactions was run in a pouch 110 of FIG. 5 , on an instrumentsimilar to instrument 800 but configured for pouch 110. To show celllysis and effectiveness of the two-stage nucleic acid amplification, 50μL each of a live culture of S. cerevisaie and S. pombe at log phase wasmixed with 100 μL of a nasopharyngeal aspirate sample from a healthydonor to form the sample, then mixed with 200 μL lysis buffer (6Mguanidine-HCl, 15% TritonX 100, 3M sodium acetate. 300 μL of the 400 μLsample in lysis buffer was then injected into chamber 192 a of pouch110.

The pouch 110 was manufactured with 0.25 g ZS beads sealed inthree-lobed blister 122. Second-stage primers, as discussed below, werealso spotted in blisters 181 and 182 during manufacture of pouch 110.The pouch 110 was loaded as follows:

-   -   115 a sample and lysis buffer, as described above,    -   115 b magnetic beads in the lysis buffer,    -   115 d-e wash buffer (10 mM sodium citrate),    -   115 g elution buffer (10 mM Tris, 0.1 mM EDTA)    -   115 h first-stage PCR buffer:        -   0.2 mM dNTPs        -   0.3 μM each primer:            -   Sc1: primers configured for amplifying a portion of the                YRA1 nuclear protein that binds to RNA and to MEX67p                of S. cerevisaie. The primers are configured to amplify                across an intron such that amplification of cDNA (mRNA                reverse-transcribed via M-MLV) yields a 180 bp amplicon.            -   Sc2: primers configured for amplifying a 121 bp region                of the cDNA of MRK1 glycogen synthase kinase 3 (GSK-3)                homolog of S. cerevisaie.            -   Sc3: primers configured for amplifying a 213 bp region                of the cDNA of RUB1 ubiquitin-like protein of S.                cerevisaie.            -   Sp1: primers configured for amplifying a 200 bp region                of the cDNA of suc1-cyclin-dependent protein kinase                regulatory subunit of S. pombe.            -   Sp2: primers configured for amplifying a 180 bp region                of the cDNA of sec14-cytosolic factor family of S.                pombe.        -   PCR buffer with 3 mM MgCl₂ (without BSA)        -   50 units M-MLV        -   4.5 units Taq:Antibody        -   100 units RNAseOut    -   115 j-k second-stage PCR buffer        -   0.2 mM dNTPs        -   1× LC Green® Plus (Idaho Technology)        -   PCR buffer with 2 mM MgCl₂ (with BSA),        -   4.5 units Taq    -   115 l second-stage PCR buffer with a sample of the first-stage        amplicons.

During manufacture, second-stage blisters 181 and 182 were spotted withnested second-stage primers. Each blister was spotted with one primerpair in an amount to result in a final concentration of about 0.3 μMonce rehydrated with the second-stage PCR buffer. The second-stagenested primers are as follows:

-   -   Sc1: primers configured for amplifying an 80 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc2: primers configured for amplifying a 121 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc3: primers configured for amplifying a 93 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp1: primers configured for amplifying a 99 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp2: primers configured for amplifying a 96 bp portion of the        Sc1 cDNA first-stage amplicon.        There is no overlap between the first-stage and second stage        primer pairs for any of the targets. Each pair of primers was        spotted into one negative control blister 181 and two        second-stage blisters 182, so that each second-stage        amplification would be run in duplicate, each duplicate with a        negative control.

After loading, activation of the plunger associated with entry channel115 a moved the sample to three-lobed blister 122, activation of theplunger associated with entry channel 115 b moved the magnetic beads toreservoir 101, activation of the plungers associated with entry channels115 d-e moved wash buffer to reservoirs 102 and 103, activation of theplunger associated with entry channel 115 g moved elution buffer toreservoir 104, activation of the plunger associated with entry channel115 h moved first-stage PCR buffer to reservoir 105, activation of theplungers associated with entry channels 115 j-k moved second stage PCRbuffer to reservoirs 106 and 107, and activation of the plungerassociated with entry channel 115 l moved the positive control(second-stage PCR buffer with a sample of previously preparedfirst-stage amplicon) to reservoir 108. In this present example, theplungers associated with entry channels 115 a and 115 b were depressedprior to loading the pouch 110 into the instrument. All other plungerswere depressed sequentially in the instrument during the run, and fluidswere moved to reservoirs 102 through 108 as needed.

Once pouch 110 was placed into the instrument, and beating took placefor ten minutes in the presence of ZS beads, as described above. Oncecell lysis was complete, reservoir 101 was compressed and nucleic acidbinding magnetic beads from reservoir 101 were forced into three-lobedblister 122, where the beads were mixed gently and allowed to incubatefor 5 minutes.

The sample-bead mixture was then moved to blister 144, where themagnetic beads were captured via activation of the magnet. Once themagnet was deployed, bladders adjacent blister 144 were pressurized toforce fluids back to three-lobed blister 122. The captured beads werethen washed as described above, using the wash solution from reservoirs102 and 103. Following washing, the beads were once again captured inblister 144 via activation of the magnet, and the elution buffer storedin reservoir 104 is moved to blister 144, where, after a 2 minuteincubation, the nucleic acids eluted from the beads are then moved toblister 161, as discussed above.

In blister 161, the nucleic acid sample is mixed with first-stage PCRmaster mix from reservoir 105. The sample is then held at 40° C. for 10minutes (during which time M-MLV converts mRNA to cDNA), then 94° C. for2 minutes (to inactivate the M-MLV and remove antibody from taq).Thermal cycling is then 20 cycles of 94° C. for 10 second and 65° C. for20 seconds.

Subsequent to first-stage amplification, the sample is dilutedapproximately 100-fold using the second-stage PCR master mix fromreservoir 106. The sample is then moved to blisters 182, which werepreviously spotted with the second-stage primers, as discussed above.Second-stage PCR buffer was moved from reservoir 181 to negative controlblisters 181, and the positive control mixture was moved to blisters 183from reservoir 108. The samples were denatured for 30 seconds at 94° C.,then amplified for 45 cycles of 94° C. for 5 seconds and 69° C. for 20seconds.

As can be seen in FIG. 13 , all target amplicons and the positivecontrol showed amplification, while none of the negative controls showedamplification. Each sample was run in replicates. The replicates eachshowed similar amplification (data not shown).

It is understood that the S. cerevisaie and S. pombe targets areillustrative only and that other targets are within the scope of thisinvention.

Example 2: iPCR

In another example, the pouches and instruments of the present inventionmay be used for immuno-PCR (iPCR). iPCR combines the antibodyspecificity of ELISA with the sensitivity and multiplex capabilities ofPCR. While iPCR has been applied to diagnostics and toxin detection,iPCR has not enjoyed widespread commercial application, presumablybecause PCR template contamination issues are severe in an open ELISAformat. Because the pouch format of the present invention provides asealed environment, the pouches of the present invention may be wellsuited for iPCR.

A traditional ELISA detection scheme is shown in FIG. 11 (labeled“ELISA”). In 1992, Cantor and colleagues (Sano, T., et al, Science,1992. 258(5079): p. 120-2, herein incorporated by reference) described amodification of the basic ELISA technique (FIG. 11 , similar to the“Immuno-PCR I” scheme without capture antibody C-Ab), in which theenzyme used for generating a specific signal is replaced by a unique DNAfragment indirectly attached to the reporter antibody R through abi-functional binding moiety S, such as a streptavidin-protein Achimera. The DNA fragment is subsequently detected by PCR. It is knownthat PCR detection can provide dramatic increases in immuno-PCR assaysensitivity over corresponding ELISA assays, with improvements tosensitivity commonly 10² to 10⁴-fold. Advances in quantitative real-timePCR methods have improved the speed and quantification of immuno-PCR.Direct coupling of the reporter antibody (R-Ab) with DNA template tags(FIG. 11 , “Immuno-PCR II” scheme) has further increased the assaysensitivity 10² to 10³ fold and made possible the development ofmultiplex immuno-PCR assays, in which each different antibody is taggedwith a different oligonucleotide and, thus, each antigen is associatedwith a unique amplification product.

Despite these advantages over traditional ELISAs, iPCR has not beenwidely adopted in commercial products in the 13 years since it was firstdescribed. This is due in part to the contamination hazards inherent inany open-tube PCR analysis method. Prior art iPCR protocols are derivedfrom ELISA assays and require numerous wash steps that increase thelikelihood of contaminating the work area with amplified material. Thesignificant risk of false positives due to workflow contamination hascontributed to the avoidance of iPCR in diagnostic assessment.

Amplicon contamination issues slowed the widespread adoption of PCRitself in the diagnosis of human genetic conditions or of infectiousdisease until homogenous (i.e. “closed-tube”) PCR assays were developed.By making the readout of the assay possible in a closed-tube system,spread of amplicon is severely curtailed. Similarly, iPCR may be morewidely adopted if a closed system format were available. In the presentsystem, the sample would be injected into a pouch that would be providedwith all required reagents. The steps of antigen capture, wash,reporter-antibody binding, wash, and subsequent PCR detection could beperformed completely within the pouch. Illustratively, nucleic acidswould never leave the pouch and would be disposed of along with thepouch.

Any of the pouches of the present invention may be adapted for iPCR. Forexample, the pouch 210 of FIG. 6 illustratively may be adapted asfollows. Chambers 292 a through 292 l would be filled with the followingcomponents. The sample illustratively comprising an unpurified and/orunmodified antigen (e.g. a toxin) is injected through injection port 241a to chamber 292 a. A capture antibody conjugated to magnetic beads(C-Ab) is provided in chamber 292 b. If multiple targets are to betested, it is understood that multiple capture antibodies havingspecificity for multiple antigens may be used. An optional pre-washbuffer is provided in chamber 292 c. A reporter antibody conjugated toan oligonucleotide template (R-Ab-DNA) is provided in chamber 292 d. Itis understood that the capture and reporter antibodies may be monoclonalor polyclonal. When multiple antigens are to be detected, the captureand reporter antibodies may contain only polyclonal antibodies, onlymonoclonal antibodies, or any combination of polyclonals specific forone antigen and monoclonals specific for another antigen. When areporter antibody is polyclonal, it is understood that all reporterantibodies having specificity for a particular antigen will be coupledto oligonucleotide templates have one specific sequence, even if thespecificity between various antibodies in that set varies. Theoligonucleotide may be double-stranded or single-stranded. MultipleR-Ab-DNAs may be provided to detect multiple antigens, with eachdifferent antibody conjugated to a unique oligonucleotide. Wash buffersare provided in chambers 292 e through 292 h. A first-stage PCR mastermix, as described above, is provided in chamber 292 i. A dilution bufferis provided in chamber 292 j. A second-stage PCR master mix, asdescribed above, is provided in chamber 292 k. As discussed above, thereagents may be provided dried in chambers 292 b through 292 l, and maybe rehydrated prior to use via injection of water through seal 239, oreach reagent may be provided wet via injection to each individualchamber 292. Combinations thereof are contemplated.

Once the sample and reagents are loaded, the pouch 220 is inserted intothe instrument 800. Plunger 268 a is then depressed and the sample ismoved to three-lobed blister 222. Plunger 268 b is also depressed andthe capture antibodies conjugated to magnetic beads (shown as C in FIG.11 ) are also moved to three-lobed blister 222. The sample and the C-Abare mixed via pressure from bladder 828 alternating with pressure frombladders 824, 826. Because mixing is desired, pressure from the bladders824, 826, 828 may be considerably lower than the pressure used asdiscussed above for lysis. Illustratively, gentle mixing is obtained.The sample and the C-Ab are allowed to incubate for sufficient time forthe capture antibodies to bind to antigens T in the sample (formingC-Ab-T complexes), illustratively for about 5 minutes, although otherincubation times may be desirable. For iPCR, it may be desirable toinclude an additional heater in instrument 800 to maintain incubationsat about 37° C.

Once the antigens present in the sample have been sufficiently incubatedfor capture, the sample is moved to blister 246 and the magnet 850 isdeployed, capturing the capture antibodies in blister 246. The unboundportions of the sample are then moved back to three-lobed blister 222,which now functions as a waste reservoir. While magnetic beads are usedto restrain the capture antibodies in the examples described herein, itis understood that other capture mechanisms may be used, including solidsupports, possibly even cross-linking the capture antibodies to aninterior surface of a blister.

If desired, the C-Ab-T complexes may be washed using the pre-wash bufferfrom chamber 292 c. Pre-wash buffer is moved into blister 244 viachannel 245, the magnet is withdrawn, releasing the C-Ab-T complexes,and the beads are gently moved between blisters 244 and 246. The beadsare then recaptured in blister 246 via activation of the magnet 850, andthe remaining fluid is moved to three-lobed blister 222. It is expectedthat this pre-wash may improve discrimination of a positive signal overthe background negative signal, but such differences may prove to beinsignificant. Additional pre-washes may be performed, if desired.

Plunger 268 d is depressed and the mixture containing one or morereporter antibodies R-Ab conjugated to oligonucleotide templates(R-Ab-DNA, shown in FIG. 11 , Immuno-PCR II scheme, as R with attachednucleic acid) is moved to blister 246. The magnet is retracted and themixture is gently mixed by moving between blisters 244 and 246.Incubation, illustratively for about 5 minutes although other incubationtimes may be desirable, allows formation of the ternary complexesC-Ab-T-R-Ab-DNA, as illustrated in FIG. 11 , Immuno-PCR II. Activationof the magnet 850 allows capture of the ternary complexes in blister246, and the remaining fluid is moved to three-lobed blister 222.

Plunger 268 e is depressed and wash buffer is moved from chamber 292 eto blister 246. The magnetic bead-ternary complex is washed as in thepre-wash described above, the magnetic bead-ternary complex isrecaptured in blister 246, and the remaining fluid is moved tothree-lobed blister 222. Washing is repeated multiple times using thewash buffers from chambers 292 f, 292 g, and 292 h, except that mixingis between blisters 246 and 248 to avoid reintroducing unbound R-Ab-DNAcomplexes that may be residing in blister 244 or channel 243. While fourwashes are described in this illustrative embodiment, it is understoodthat any number of washes may be used, illustratively by altering thenumber of chambers in the fitment 290 or by increasing the volume of thechambers and using only a portion of the wash buffer in a chamber foreach wash. It is also understood that removal of all unbound R-Ab-DNAcomplexes is extremely difficult, even with a large number of washes.Further, for an antigen that is not present in the sample, the presenceof just a few molecules of unbound R-Ab-DNA or non-specifically boundR-Ab-DNA complexes specific for that antigen may result in anamplification signal. Thus, while the ideal goal of the washing step isto remove all R-Ab-DNA complexes specific for antigens that are notpresent in the sample, one illustrative goal is to remove a sufficientnumber of such R-Ab-DNA complexes such that the amplification curve forthat oligonucleotide is delayed and can be distinguished from theamplification curve of a positive sample. Illustratively, more washesshould remove more unbound R-Ab-DNA and provide for a lower detectionlimit, but more washes risk loss of desired ternary complexes throughdissociation or loss of magnetic beads not captured by the magnet. Afterwashing is complete, if desired, the captured ternary complex may beheated or enzymatically treated (illustratively with papain, proteinaseK, or other suitable enzyme provided via an additional chamber) torelease the DNA prior to PCR. Such treatment may improve the first-stagePCR efficiency. It is understood that such treatment may be used withany of the iPCR examples discussed herein.

Once washing is complete, plunger 268 i is depressed and the first-stagePCR master mix, as described above, is moved to blister 246. First-stagePCR master mix contains primer pairs for all desired targets. The magnet850 is released, and optional mixing between blisters 246 and 248 may beused to resuspend the ternary complexes. The mixture is moved to blister264, where first-stage thermal cycling takes place, as described above.Once the complexed oligonucleotides have been amplified to sufficientlevels, as discussed above, the amplified mixture is optionally dilutedusing the dilution buffer provided in chamber 292 j. Some or all of thefirst-stage amplified mixture may be mixed with the second-stage PCRmaster mix provided from chamber 292 k, and then this mixture is movedto the 18 second-stage blisters 282, where second-stage primers areprovided, as discussed above. If desired, one of the second-stageblisters 282 may be used for a negative control, wherein it is knownthat no antigen is present in the sample, but R-Ab-DNA was provided fromchamber 292 d and the proper primers are provided in the negativecontrol second-stage blister 282. It is expected that, despite variouswashes, small amounts of this particular R-Ab-DNA may be present in thefirst-stage PCR and, accordingly, that small amounts of the first-stageamplified product may be provided to this second-stage blister 282.However, the amounts should be quite small, and the crossing pointshould be delayed well past that of positive samples. Also, if desired,one of one of the second-stage blisters may be used for a positivecontrol, wherein the sample is spiked with an antigen that is nototherwise being tested (perhaps included with the C-Ab beads), whichpresumably will bind its corresponding R-Ab-DNA, and which is thenamplified in the first-stage PCR. Finally, control blisters 283 are notused in this illustrative embodiment. However, with a minorreconfiguration, blisters 283 may be connected to blister 266 and mayprovide for six additional second-stage reactions. Alternatively,blisters 283 may be used for other controls, as are desired by theparticular application.

As discussed above, because of the difficulty in removing all unbound ornon-specifically bound R-Ab-DNA complexes, even negative samples mayshow some amplification. It is expected that real-time amplificationanalysis will allow positives to be distinguished from negatives via adifference in cycle number of a threshold crossing point (or anequivalent cycle threshold measurement, such as the cycle number when50% of amplification is reached).

It is understood that the first-stage multiplex amplification may not benecessary for detection with iPCR, even when testing for multipleantigens. However, the first-stage multiplex amplification may affordmore sensitivity.

Example 3: iPCR with iPCR-Specific Pouch

The above example illustrates a method adapting the pouch 210 of FIG. 6for iPCR. However, FIG. 12 shows a pouch 310 that is illustrativelyconfigured for iPCR. Fitment 390 is similar to fitments 190 and 290,except having 15 chambers 392 and plungers 368. Each chamber 392(illustratively chamber 392 a, where the sample is injected) may haveits own injection port, or several chambers may have a connectingchannel and may share an injection port (illustratively 392 e through392 k, each containing wash buffer). As with the above-describedfitments, any combination of injection ports and channels is within thescope of this invention. Pouch 310 differs from pouch 210 of FIG. 6 inone primary way. As cell lysis is usually not needed in iPCR, thethree-lobed blister 222 may be replaced by a single large wastereservoir 322. Because multiple washes are desirable in iPCR, wastereservoir 322 is provided with a sufficiently large volume to retain themultiple used buffers, for example 2-5 ml, depending on the applicationand volume of the reactions. It is understood that instrument 800 mayneed to be reconfigured somewhat to accommodate pouch 390.

Prior to insertion into the instrument, pouch 390 of FIG. 12illustratively would have the following components in the chambers 392.The sample to be tested would be injected into chamber 392 a. Captureantibodies (C-Ab) conjugated to magnetic beads are provided in chamber392 b. An optional pre-wash buffer is provided in chamber 392 c.Reporter antibodies conjugated to their respective oligonucleotidetemplates (R-Ab-DNA) are provided in chamber 392 d. As discussed above,multiple R-Ab-DNAs may be provided to detect multiple antigens, witheach different antibody conjugated to a unique oligonucleotide. Washbuffers are provided in chambers 392 e through 392 k. A first-stage PCRmaster mix is provided in chamber 3921. A dilution buffer is provided inchambers 392 m and 392 n. A second-stage PCR master mix is provided inchamber 392 o.

To begin, plungers 368 a and 368 b are depressed, forcing the sample andthe capture antibodies C-Ab through channel 343 into blister 344. Thesample and the C-Ab are gently mixed, illustratively by moving betweenblisters 344 and 346 via channel 345, and are incubated as describedabove. After a sufficient period of time for formation of the C-Ab-Tcomplex, the mixture is moved to blister 346 via channel 338, where amagnet 350 housed in the instrument is deployed, capturing the complexedbeads therein. The remaining fluid is moved to waste reservoir 322, viachannel 339. Optionally, pre-wash buffer from chamber 392 c is moved toblister 346 via channel 345, the magnet 350 is withdrawn, and themagnetic beads are gently washed by moving the fluid between blisters344 and 346. The magnet 350 is again deployed and the beads are againcaptured in blister 346.

Next, plunger 368 d is depressed moving the reporter antibodiesconjugated to nucleic acid template (R-Ab-DNA) to blister 346, themagnet 350 is withdrawn, and the C-Ab-T and the R-Ab-DNA are gentlymixed illustratively by moving between blisters 344 and 346 via channel345 and are incubated as described above. After formation of the ternarycomplex (C-Ab-T-R-Ab-DNA), the magnet 350 is once again deployed,capturing the ternary complex in blister 346, and the remaining fluid ismoved to waste blister 322.

The ternary complex is then washed using the wash buffer from chamber392 e, as described above for the pre-wash. The magnet 350 is againdeployed, capturing the ternary complex in blister 346, and theremaining fluid is moved to waste blister 322. Washing is repeatedvarious times, using the wash buffer from chambers 392 f through 392 k.Thus, in the illustrative embodiment of FIG. 12 , seven washes arecompleted. However, as discussed above, more or fewer washes may bedesirable, depending on the particular application.

As illustrated in the Immuno-PCR II scheme shown in FIG. 11 , thereporter antibody is conjugated directly to the nucleic acid template.It is understood that the reporter antibody in any of the embodimentsdiscussed herein could be attached to the nucleic acid template by anyof a variety of ways, including direct and indirect covalent andnon-covalent bonding. Also, the reporter antibody could be attached tothe nucleic acid through a variety of mechanisms, including, forexample, through the use of secondary antibodies, as illustrated in theImmuno-PCR I scheme of FIG. 11 . If secondary antibodies or otherindirect coupling mechanisms are used, it may be desirable to addadditional ports and further washing steps.

The first-stage PCR master mix, as described above, is then deployed toblister 346 via activation of plunger 368 k, and the magnet 350 is onceagain withdrawn. If gentle mixing is desired, the fluid may be movedbetween blisters 346 and 364 via channel 347. While mixing can takeplace between blisters 346 and 344 as before, in the illustrativeembodiment mixing takes place between blisters 346 and 364. This aids inreducing the reintroduction of unbound reporter antibody complexes thatmay be residing in blister 344. The sample is then moved to blister 364.A bladder positioned over 364 is gently pressurized to move blister 364into contact with a heating/cooling device, such as a Peltier device,and the sample would be thermocycled, as discussed above for first-stagePCR. As discussed above in the previous example, first-stage PCR may beunnecessary with the presently described iPCR, blister 364 and itsassociated heater may be omitted, and all washes illustratively couldtake place by mixing between blisters 344 and 346. If first-stage PCR isomitted, the dilution, as discussed below may also be omitted.

Most of the amplified sample is moved to waste blister 322, leaving someamplified sample behind in blister 364 to be diluted. It is understoodthat if space constraints or other considerations limit the size ofblister 322, blisters 344 and 346 may be used to contain the remainingwaste. The small amount of remaining amplified sample is mixed withdilution buffer from chamber 392 m, which has been moved to blister 366via channel 349. The sample and the dilution buffer may be mixed gentlybetween blisters 364 and 366, via channel 355. If further dilution isdesired, dilution may be repeated using the dilution buffer from chamber392 n. Finally, some of the diluted sample is moved to waste reservoir322 and the remaining diluted sample is mixed with second-stage PCRmaster mix from chamber 392 o. After mixing, the sample is moved to thevarious low volume second stage blisters 382, where second-stage primersare provided, as discussed above. In the present configuration, blister383 may be used for a negative control and blister 384 may be used for apositive control, as discussed above in the previous iPCR example.Second-stage PCR and analysis takes place as described above in theprevious iPCR example.

Example 4 Combined PCR and iPCR

In some circumstances, it may be desirable to test for antigens andnucleic acids in one reaction set. For example, a terrorist attack mayemploy various agents to kill multiple people. In responding to theattack, it may be unknown if the causative agent is a virus, bacterium,or other organism, or if the causative agent is a toxin. Theclosed-environment system of the pouches of the present invention iswell suited for such use. In the embodiment disclosed herein, both PCRand iPCR may take place within a single pouch, allowing for simultaneousdetection of various biological and antigenic agents.

FIG. 13 shows a pouch 410 that is similar to pouch 210 of FIG. 6 .Illustrative pouch 410 has all of the blisters of pouch 210, but alsoincludes blisters 430, 431, 432, and 433. Pouch 410 also has a largerfitment 490, having twenty chambers 492 with twenty correspondingplungers 468. As above, the fitment could include separate injectionports for each chamber, or various chambers could have connectingchannels. Various combinations thereof are within the scope of thisinvention. The instrument for pouch 410 would be similar to instrument800, except that additional pneumatic actuators would be needed forblisters 430, 431, 432, and 433 and channels 436, 457, 473, 486, 487,and 488, as well as two additional retractable magnets 451 and 454adjacent blisters 433 and 431, respectively.

In the illustrative embodiment, the chambers would be loaded as follows.iPCR wash buffer would be provided in chambers 492 a through 492 e and492 j. The sample to be tested would be injected into chamber 492 f Thecapture antibodies (C-Ab) conjugated to magnetic beads are provided inchamber 492 g. An optional pre-wash buffer is provided in chamber 492 h.Reporter antibodies conjugated to their respective oligonucleotidetemplate (R-Ab-DNA) are provided in chamber 492 i. A cell lysis bufferis provided in chamber 492 k. Nucleic-acid-binding magnetic beads areprovided in chamber 492 l. Nucleic acid wash buffers are provided inchambers 492 m and 492 n. A nucleic acid elution buffer is provided inchamber 492 o. A first-stage PCR master mix is provided in chamber 492p. A dilution buffer is provided in chambers 492 q and 492 r. Asecond-stage PCR master mix is provided in chamber 492 s. Controls, asdiscussed above with respect to FIG. 6 , are provided in chamber 492 t.It is understood that this arrangement is illustrative and that otherconfigurations are possible. Also, as with the other examples discussedabove, one or more of these components may be provided dried in one ormore of the blisters of pouch 410.

Once the sample is loaded into chamber 492 f and pouch 410 is loadedinto the instrument, plungers 468 f and 468 g are depressed, moving thesample and C-Ab through channel 436 to blister 430. The sample andcapture antibodies may be mixed by gently moving them between blisters430 and 431 and then incubated as described above, to encourageformation of C-Ab-T complexes. The sample is moved to blister 431 andmagnet 454 is activated, capturing the C-Ab-T complexes therein. Thus,toxins or other targeted antigens are now captured in blister 431. It isnoted that, in the illustrative embodiment, the surface of the magneticbead portion of the magnetic beads coupled to the capture antibodies isdifferent from the surface of the nucleic-acid-binding magnetic beads,and the magnetic beads coupled to the capture antibodies isillustratively configured not to bind nucleic acids. The remaining fluidis then moved to three-lobed blister 422 via channel 473. This fluid canthen be processed and assayed for the presence of target nucleic acids.This division of the sample may be problematic if a targeted antigen isa surface antigen of an organism targeted in the PCR detection. In sucha situation, it may be desirable to choose between antigen detection andnucleic acid detection for that organism, or to use separate pouches forPCR and iPCR. Alternatively, the sample may be lysed prior to antibodycapture. If lysis would interfere with antibody capture, for example bychanging the conformation of the antigen, then the sample may be dividedand just a portion of the sample may be lysed prior to antibody capture.If a pre-wash of the C-Ab-T is desired, plunger 468 h is activated andthe pre-wash buffer from chamber 492 h is moved into blister 431. Magnet454 is withdrawn, the fluid is mixed between blisters 430 and 431, andmagnet 454 is once again deployed, capturing the C-Ab-T complex inblister 431. The wash buffer, now possibly containing cells that hadbeen left behind after capture, is moved to three-lobed blister 422,along with the rest of the uncaptured material.

It is understood that the sample is now divided into two parts forseparate processing. Antigens present in the sample are now captured inC-Ab-T complexes in blister 431, while cells, viruses, and free nucleicacids present in the sample are now in three-lobed blister 422 awaitinglysis. The two portions of the sample are processed separately untilboth are ready for first-stage PCR. These processes may take place inany order or simultaneously. However, in the present embodiment, celllysis must take place prior to substantial processing of the C-Ab-Tcomplexes, so that three-lobed blister may then function as the wastereservoir. If a separate waste reservoir is used, cell lysis can bedelayed until after the C-Ab-T complexes have been processed, ifdesired.

Lysis buffer from chamber 492 k is moved into three-lobed blister 422via channel 436. Bladders adjacent the blisters of three-lobed blister422 are pressurized as described above with respect to FIG. 6 , drivinghigh velocity collisions, shearing the sample, and liberating nucleicacids. Once the cells have been adequately lysed, plunger 468 l isactivated and nucleic acid binding magnetic beads stored in chamber 492l are injected via channel 436 into three-lobed blister 220. The sampleis mixed with the magnetic beads and the mixture is allowed to incubate.The processing then continues as described above with respect to thepouch of FIG. 6 . The mixture of sample and beads are forced throughchannel 438 into blister 444, then through channel 443 and into blister446, where a retractable magnet 450 captures the magnetic beads from thesolution. The un-captured liquid is then forced out of blister 446 andback through blister 444 and into blister 422, which is now used as awaste receptacle. Plunger 468 m may be activated to provide a washsolution to blister 444 via channel 445, and then to blister 446 viachannel 447. Magnet 450 is retracted and the magnetic beads are washedby moving the beads back and forth from blisters 444 and 446. Once themagnetic beads are washed, the magnetic beads are recaptured in blister446 by activation of magnet 450, and the wash solution is then moved toblister 422. This process may be repeated using wash reagents inchambers 492 n. However, it is understood that more or fewer washes arewithin the scope of this invention. After washing, elution buffer storedin chamber 492 o is moved via channel 447 to blister 448, and the magnet450 is retracted. The solution is cycled between blisters 446 and 448via channel 452, breaking up the pellet of magnetic beads in blister 446and allowing the captured nucleic acids to come into solution. Themagnet 450 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is moved into blister448.

Returning back to blister 431, the C-Ab-T complexes are thereincaptured. Plunger 468 i is depressed and the reporter antibodiesconjugated to nucleic acid template (R-Ab-DNA) are introduced to blister430, the magnet 454 is withdrawn, and the C-Ab-T and the R-Ab-DNA aregently mixed, illustratively by moving between blisters 430 and 431 viachannel 457, and are incubated as described above. After formation ofthe ternary complex (C-Ab-T-R-Ab-DNA), magnet 454 is once againdeployed, capturing the ternary complex in blister 431, and theremaining fluid is moved to blister 422, which is now used as a wastereservoir.

The ternary complex is then washed using the wash buffer from chamber492 j, as described above for the pre-wash. Magnet 454 is againdeployed, capturing the ternary complex in blister 446, and theremaining fluid is moved to blister 422. Additional wash buffer fromchamber 492 a is injected into blister 432 via channel 486, the magnet454 is withdrawn, and the ternary complex is resuspended by mixing thefluids blisters 431 and 432. The fluids are then moved to blister 433via channel 487 and the ternary complex is captured therein viaactivation of magnet 451. The waste fluids are then moved back throughblisters 433 and 432 to blister 422. Additional wash buffer isintroduced into blister 432 from chamber 492 b and washing is repeatedby mixing between blisters 432 and 433. Washing is repeated varioustimes using the wash buffer from chambers 492 c through 492 e. Thus, inthe illustrative embodiment of FIG. 13 , six washes are completed.However, as discussed above, more or fewer washes may be desirable,depending on the particular application. It is understood that blisters432 and 433 are used to minimize contamination from prior washes. Ifdesired, blisters 432 and 433 may be omitted and the wash bufferscontained in chambers 492 a through 492 e may be provided directly toeither blister 430 or 431, with mixing between blisters 430 and 431.

The washed antibody ternary complex is now captured in blister 433 andthe eluted nucleic acids are now in blister 448. It is noted that theantibody ternary complex and the eluted nucleic acids may be processedthrough PCR in independent reactions, through to separate sets ofsecond-stage PCR blisters. However, in the present embodiment theantibody ternary complex and the eluted nucleic acids are combined forPCR analysis. First-stage PCR master mix, containing all first-stageprimers, is injected from chamber 492 p into blister 448. The nucleicacid sample is then mixed between blisters 448 and 464 via channel 453.If first-stage PCR is desired for the iPCR components, the nucleic acidsample is then moved to blister 433, magnet 451 is withdrawn, and there-united sample is illustratively mixed between blisters 433 and 464.The sample is then moved to blister 464, where the sample isthermocycled, as discussed above. Next, the amplified sample may bediluted once or several times, using the dilution buffers from chambers492 q and 492 r. Prior to each dilution, a large portion of theamplified sample is removed from blister 464 via either channel 447 orchannel 488. With each addition of dilution buffer, the sample is mixedbetween blisters 464 and 466 via channel 462. After dilution, all or aportion of the sample is mixed with the second-stage PCR master mix fromchamber 492 s, as described in the examples above.

The sample is then moved from blister 466 via channel 465 to blisters482 in second-stage amplification zone 480. Blisters 482 each had beenpreviously provided with a primer pair, some of the primer pairsspecific for target nucleic acids, while other primer pairs specific foran oligonucleotide conjugated to a reporter antibody. If desired, twoblisters 482 may be dedicated to iPCR controls, as discussed above.Blisters 483 may be used for PCR controls, as discussed above withrespect to blisters 283 of FIG. 6 . While 18 blisters 482 are shown, itis understood that any number of blisters 482 may be used. Second-stagePCR amplification proceeds as discussed above with respect to FIG. 6 .It is understood that PCR analysis may use amplification curves, meltingcurves, or a combination thereof, while iPCR analysis may use crossingthresholds, as discussed above. Other methods of analysis are within thescope of this invention.

REFERENCES

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Wittwer C T, Reed G H, Gundry C N, Vandersteen J G, Pryor R J.,    High-resolution genotyping by amplicon melting analysis using    LCGreen. Clin Chem. 2003 June; 49(6 Pt 1):853-60.-   7. McKinney J T, Longo N, Hahn S, Matern D, Rinaldo P, Dobrowolski    S F. Comprehensive analysis of the human medium chain acyl-CoA    dehydrogenase gene. Mol Gen Metab. In press-   8. Dobrowolski S F, Amat di San Filippo C, McKinney J T, Wilcken B,    Longo N Identification of novel mutations in the SLC22A5 gene in    primary carnitine deficiency with dye-binding/high-resolution    thermal denaturation, Human Mutation, submitted-   9. McKinney J T, Saunders C, Dobrowolski S F, High-resolution    melting analysis of the human galactose-1-phosphate uridyl    transferase gene, in preparation-   10. http://www.defenselink.mil/contracts/2003/ct20030925.html-   11. 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Cantor, Immuno-PCR: very    sensitive antigen detection by means of specific antibody-DNA    conjugates. Science, 1992. 258(5079): p. 120-2.-   17. Niemeyer, C. M., M. Adler, and R. Wacker, Immuno-PCR: high    sensitivity detection of proteins by nucleic acid amplification.    Trends Biotechnol, 2005. 23(4): p. 208-16.-   18. Adler, M., Immuno-PCR as a clinical laboratory tool. Adv Clin    Chem, 2005. 39: p. 239-92.-   19. Barletta, J. M., et al., Detection of ultra-low levels of    pathologic prion protein in scrapie infected hamster brain    homogenates using real-time immuno-PCR. J Virol Methods, 2005.    127(2): p. 154-64.-   20. Adler, M., et al., Detection of Rotavirus from stool samples    using a standardized immuno-PCR (“Imperacer”) method with end-point    and real-time detection. Biochem Biophys Res Commun, 2005.    333(4): p. 1289-94.-   21. Lind, K. and M. Kubista, Development and evaluation of three    real-time immuno-PCR assemblages for quantification of PSA. 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While references are made herein to PCR and iPCR, it is understood thatthe devices and methods disclosed herein may be suitable for use withother nucleic acid amplification or other biological processing methods,as are known in the art, particularly methods that benefit from afirst-stage multiplex reaction and a second-stage individual reaction.Illustrative non-limiting second-stage reactions include primerextension, including allele-specific primer extension; extensionterminations, including termination by incorporation of one or moredideoxy nucleotides; incorporation of fluorescent or non-fluorescentlabels; and other enzymatic reactions requiring a change in reactionmixture components or component ratios, such as asymmetric PCR,allele-specific PCR, invader assays, and other isothermal amplificationor detection chemistries.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

The invention claimed is:
 1. A method of nucleic acid extraction andmultiplex PCR in a self-contained system, comprising: (a) providing theself-contained system having, in fluid communication: i. one or moreports, including an injector port for introducing a sample into theself-contained system, wherein the one or more ports are sealable portsthat provide the only access from an exterior of the self-containedsystem such that when all of the one or more ports are closed, theself-contained system is a fully closed, sealed environment thatprevents access from the self-contained system to surroundingatmosphere, ii. a cell lysis zone configured for lysing cells or sporeslocated in the sample, iii. a nucleic acid preparation zone, the nucleicacid preparation zone configured for purifying a plurality of nucleicacids that may be in the sample, wherein the nucleic acid preparationzone is fluidly connected to the injector port through the cell lysiszone; iv. at least one amplification zone, the amplification zoneconfigured for amplification of the plurality of nucleic acids that maybe in the sample; (b) introducing the sample into the self-containedsystem via the injector port; (c) lysing cells or spores in the celllysis zone by impacting the cell lysis zone with rotating blades orpaddles for bead-milling; (d) preparing the plurality of nucleic acidsthat may be in the sample in the nucleic acid preparation zonesubsequent to step (c); (e) moving the plurality of nucleic acids thatmay be in the sample into the amplification zone to mix with a pluralityof primer pairs in the amplification zone; (f) thermal cycling theplurality of nucleic acids that may be in the sample in theamplification zone in the presence of PCR reaction components and theplurality of primer pairs to create an amplification mixture; (g)detecting which of the plurality of nucleic acids are present in theamplification mixture in the amplification zone; and (h) closing the oneor more ports, wherein step (h) takes place after step (b).
 2. Themethod of claim 1, wherein at least one of the ports of the one or moreports is a one-way valve.
 3. The method of claim 2, wherein theamplification zone is provided with dried amplification reagentstherein.
 4. The method of claim 1, wherein the amplification zone isprovided with dried amplification reagents therein.
 5. The method ofclaim 1, wherein the detecting step includes detecting fluorescentemission from a fluorescent dye in the amplification zone.
 6. The methodof claim 5, wherein the fluorescent dye is a dsDNA binding dye.
 7. Themethod of claim 5, wherein the fluorescent dye is incorporated in afluorescently labeled oligonucleotide probe.
 8. The method of claim 1,wherein each of the plurality of primer pairs is configured to amplifynucleic acids from a different species.